Dry powder formulations and methods for treating pulmonary diseases

ABSTRACT

The present invention is directed toward respirable dry particles for delivery of divalent metal cation salts and/or monovalent cation salts to the respiratory tract and methods for treating a subject having a respiratory disease and/or infection.

RELATED APPLICATIONS

This application is a continuation if U.S. patent application Ser. No.14/667,857 filed on Mar. 25, 2015, which is a continuation of U.S.patent application Ser. No. 14/456,445 filed on Aug. 11, 2014, which isa continuation of U.S. patent application Ser. No. 13/259,635, filed onNov. 21, 2011, which is the U.S. National Stage of InternationalApplication No. PCT/US2010/028961, filed Mar. 26, 2010, published inEnglish, and claims the benefit of U.S. Provisional Application No.61/305,819, filed on Feb. 18, 2010, U.S. Provisional Application No.61/298,092, filed on Jan. 25, 2010, U.S. Provisional Application No.61/267,747, filed on Dec. 8, 2009, U.S. Provisional Application No.61/255,764, filed on Oct. 28, 2009, U.S. Provisional Application No.61/163,772, filed on Mar. 26, 2009, U.S. Provisional Application No.61/163,767, filed on Mar. 26, 2009 and U.S. Provisional Application No.61/163,763, filed on Mar. 26, 2009. The entire contents of each of theforegoing applications are incorporated herein by reference.

BACKGROUND OF THE INVENTION

Pulmonary delivery of therapeutic agents can offer several advantagesover other modes of delivery. These advantages include rapid onset, theconvenience of patient self-administration, the potential for reduceddrug side-effects, ease of delivery by inhalation, the elimination ofneedles, and the like. Inhalation therapy is capable of providing a drugdelivery system that is easy to use in an inpatient or outpatientsetting, results in very rapid onset of drug action, and producesminimal side effects.

Metered dose inhalers (MDIs) are used to deliver therapeutic agents tothe respiratory tract. MDIs are generally suitable for administeringtherapeutic agents that can be formulated as solid respirable dryparticles in a volatile liquid under pressure. Opening of a valvereleases the suspension at relatively high velocity. The liquid thenvolatilizes, leaving behind a fast-moving aerosol of dry particles thatcontain the therapeutic agent. MDIs are reliable for drug delivery onlyto mid-sized airways for the treatment of respiratory ailments. However,it is the small-sized airways (i.e., bronchioles and alveoli) that areoften the site of manifestation of pulmonary diseases such as asthma andinfections.

Liquid aerosol delivery is one of the oldest forms of pulmonary drugdelivery. Typically, liquid aerosols are created by an air jetnebulizer, which releases compressed air from a small orifice at highvelocity, resulting in low pressure at the exit region due to theBernoulli effect. See, e.g., U.S. Pat. No. 5,511,726. The low pressureis used to draw the fluid to be aerosolized out of a second tube. Thisfluid breaks into small droplets as it accelerates in the air stream.Disadvantages of this standard nebulizer design include relatively largeprimary liquid aerosol droplet size often requiring impaction of theprimary droplet onto a baffle to generate secondary splash droplets ofrespirable sizes, lack of liquid aerosol droplet size uniformity,significant recirculation of the bulk drug solution, and low densitiesof small respirable liquid aerosol droplets in the inhaled air.

Ultrasonic nebulizers use flat or concave piezoelectric disks submergedbelow a liquid reservoir to resonate the surface of the liquidreservoir, forming a liquid cone which sheds aerosol particles from itssurface (U.S. 2006/0249144 and U.S. Pat. No. 5,551,416). Since noairflow is required in the aerosolization process, high aerosolconcentrations can be achieved, however the piezoelectric components arerelatively expensive to produce and are inefficient at aerosolizingsuspensions, requiring active drug to be dissolved at low concentrationsin water or saline solutions. Newer liquid aerosol technologies involvegenerating smaller and more uniform liquid respirable dry particles bypassing the liquid to be aerosolized through micron-sized holes. See,e.g., U.S. Pat. No. 6,131,570; U.S. Pat. No. 5,724,957; and U.S. Pat.No. 6,098,620. Disadvantages of this technique include relativelyexpensive piezoelectric and fine mesh components as well as fouling ofthe holes from residual salts and from solid suspensions.

Dry powder inhalation has historically relied on lactose blending toallow for the dosing of particles that are small enough to be inhaled,but aren't dispersible enough on their own. This process is known to beinefficient and to not work for some drugs. Several groups have tried toimprove on these shortcomings by developing dry powder inhaler (DPI)formulations that are respirable and dispersible and thus do not requirelactose blending. Dry powder formulations for inhalation therapy aredescribed in U.S. Pat. No. 5,993,805 to Sutton et al.; U.S. Pat. No.6,921,6527 to Platz et al.; WO 0000176 to Robinson et al.; WO 9916419 toTarara et al.; WO 0000215 to Bot et al; U.S. Pat. No. 5,855,913 to Haneset al.; and U.S. Pat. Nos. 6,136,295 and 5,874,064 to Edwards et al.

Broad clinical application of dry powder inhalation delivery has beenlimited by difficulties in generating dry powders of appropriateparticle size, particle density, and dispersibility, in keeping the drypowder stored in a dry state, and in developing a convenient, hand-helddevice that effectively disperses the respirable dry particles to beinhaled in air. In addition, the particle size of dry powders forinhalation delivery is inherently limited by the fact that smallerrespirable dry particles are harder to disperse in air. Dry powderformulations, while offering advantages over cumbersome liquid dosageforms and propellant-driven formulations, are prone to aggregation andlow flowability which considerably diminish dispersibility and theefficiency of dry powder-based inhalation therapies. For example,interparticular Van der Waals interactions and capillary condensationeffects are known to contribute to aggregation of dry particles. Hickey,A. et al., “Factors Influencing the Dispersion of Dry Powders asAerosols”, Pharmaceutical Technology, August, 1994.

To overcome interparticle adhesive forces, Batycky et al. in U.S. Pat.No. 7,182,961 teach production of so called “aerodynamically lightrespirable particles,” which have a volume median geometric diameter(VMGD) of greater than 5 microns (um) as measured using a laserdiffraction instrument such as HELOS (manufactured by Sympatec,Princeton, N.J.). See Batycky et al., column 7, lines 42-65. Anotherapproach to improve dispersibility of respirable particles of averageparticle size of less than 10 μm, involves the addition of a watersoluble polypeptide or addition of suitable excipients (including aminoacid excipients such as leucine) in an amount of 50% to 99.9% by weightof the total composition. Eljamal et al., U.S. Pat. No. 6,582,729,column 4, lines 12-19 and column 5, line 55 to column 6, line 31.However, this approach reduces the amount of active agent that can bedelivered using a fixed amount of powder. Therefore, an increased amountof dry powder is required to achieve the intended therapeutic results,for example, multiple inhalations and/or frequent administration may berequired. Still other approaches involve the use of devices that applymechanical forces, such as pressure from compressed gasses, to the smallparticles to disrupt interparticular adhesion during or just prior toadministration. See, e.g., U.S. Pat. No. 7,601,336 to Lewis et al., U.S.Pat. No. 6,737,044 to Dickinson et al., U.S. Pat. No. 6,546,928 toAshurst et al., or U.S. Pat. Applications 20090208582 to Johnston et al.

A further limitation that is shared by each of the above methods is thatthe aerosols produced typically include substantial quantities of inertcarriers, solvents, emulsifiers, propellants, and other non-drugmaterial. In general, the large quantities of non-drug material arerequired for effective formation of respirable dry particles smallenough for alveolar delivery (e.g. less than 5 microns and preferablyless than 3 microns). However, these amounts of non-drug material alsoserve to reduce the purity and amount of active drug substance that canbe delivered. Thus, these methods remain substantially incapable ofintroducing large active drug dosages accurately to a patient forsystemic delivery.

Therefore, there remains a need for the formation of small particle sizeaerosols that are highly dispersible. In addition, methods that produceaerosols comprising greater quantities of drug and lesser quantities ofnon-drug material are needed. Finally, a method that allows a patient toadminister a unit dosage rapidly with one or two, small volume breathsis needed.

SUMMARY OF THE INVENTION

The invention relates to respirable dry powders comprised of dryparticles that contain one or more divalent metal cations, such ascalcium (Ca²⁺), as an active ingredient, and to dry powders that containthe respirable particles. The invention also relates to respirable dryparticles that contain one or more monovalent cations (such as Na+) andto dry powders that contain the respirable particles. The activeingredient (e.g., calcium ion) is generally present in the dry powdersand dry particles in the form of one or more salts, which canindependently be crystalline, amorphous or a combination of crystallineand amorphous. The dry powders and dry particles can optionally includeadditional monovalent salts (e.g. sodium salts), therapeutically activeagents or pharmaceutically acceptable excipients. In one aspect, therespirable dry particles may be small and highly dispersible. In anotheraspect, the respirable dry particles may be large or small, e.g., ageometric diameter (VMGD) between 0.5 microns and 30 microns.Optionally, the MMAD of the particles may be between 0.5 and 10 microns,more preferably between 1 and 5 microns.

In some aspects, the respirable dry powders have a volume mediangeometric diameter (VMGD) of about 10 microns or less and adispersibility ratio [ratio of VMGD measured at dispersion pressure of 1bar to VMGD measured at 4 bar] (1/4 bar) of less than about 2 asmeasured by laser diffraction (RODOS/HELOS system), and contain acalcium salt; that provides divalent metal cation in an amount of about5% or more by weight of the dry powder. The respirable dry powders canfurther comprise a monovalent salt that provides monovalent cation, suchas Na⁺, in an amount of about 6% or more by weight of the powders.

The respirable dry powders can have a Fine Particle Fraction (FPF) ofless than 5.6 microns of at least 45%, FPF of less than 3.4 microns ofat least 30%, and/or FPF of less than 5.0 microns of at least 45%.Alternatively or in addition, the respirable dry powders can have a massmedian aerodynamic diameter (MMAD) of about 5 microns or less. Themolecular weight ratio of divalent metal cation to the divalent metalcation salt contained in the respirable dry particle can be greater thanabout 0.1 and/or greater than about 0.16.

The respirable dry powder compositions can include a pharmaceuticallyacceptable excipient, such as leucine, maltodextrin or mannitol, whichcan be present in an amount of about 50% or less by weight, preferablyin an amount of about 20% or less by weight.

The divalent metal cation salt present in the respirable dry powders canbe a beryllium salt, a magnesium salt, a calcium salt, a strontium salt,a barium salt, a radium salt and a ferrous salt. For example, thedivalent metal cation salt can be a calcium salt, such as calciumlactate, calcium sulfate, calcium citrate, calcium chloride or anycombination thereof. The monovalent salt that is optionally present inthe respirable dry particle can be a sodium salt, a lithium salt apotassium salt or any combination thereof.

In certain aspects, the respirable dry powder contains a divalent metalcation salt and a monovalent salt, and contains an amorphous divalentmetal cation phase and a crystalline monovalent salt phase. The glasstransition temperature of the amorphous phase can be least about 120° C.These respirable dry particles can optionally contain an excipient, suchas leucine, maltodextrin and mannitol, which can be amorphous,crystalline or a mixture of forms. The respirable dry particle can havea heat of solution between about −10 kcal/mol and 10 kcal/mol.

Preferably, the divalent metal cation salt is a calcium salt, and themonovalent salt is a sodium salt. The calcium salt can be calciumcitrate, calcium lactate, calcium sulfate, calcium chloride or anycombination thereof, and the sodium salt can be sodium chloride.

In other aspects, the respirable dry powder contains a divalent metalsalt that provides a cation in an amount of about 5% or more by weightof the dry powder, the respirable dry powder have a Hausner Ratio ofgreater than 1.5 and a 1/4 bar or 0.5/4 bar of 2 or less.

The invention also relates to a respirable dry powder that containsrespirable dry particles that contain calcium citrate or calciumsulfate, and that are made using a process that includes a) providing afirst liquid feed stock comprising an aqueous solution of calciumchloride, and a second liquid feed stock comprising an aqueous solutionof sodium sulfate or sodium citrate; b) mixing the first liquid feedstock and the second liquid feed stock to produce a mixture in which ananion exchange reaction occurs to produce a saturated or supersaturatedsolution comprising calcium sulfate and sodium chloride, or calciumcitrate and sodium chloride; and c) spray drying the saturated orsupersaturated solution produced in b) to produce respirable dryparticles. Mixing in b) can be batch mixing or static mixing.

The invention also relates to methods for treating a respiratorydisease, such as asthma, airway hyperresponsiveness, seasonal allergicallergy, bronchiectasis, chronic bronchitis, emphysema, chronicobstructive pulmonary disease, cystic fibrosis and the like, comprisingadministering to the respiratory tract of a subject in need thereof aneffective amount of the respirable dry particles or dry powder. Theinvention also relates to methods for the treatment or prevention ofacute exacerbations of chronic pulmonary diseases, such as asthma,airway hyperresponsiveness, seasonal allergic allergy, bronchiectasis,chronic bronchitis, emphysema, chronic obstructive pulmonary disease,cystic fibrosis and the like, comprising administering to therespiratory tract of a subject in need thereof an effective amount ofthe respirable dry particles or dry powder.

The invention also relates to methods for treating, preventing and/orreducing contagion of an infectious disease of the respiratory tract,comprising administering to the respiratory tract of a subject in needthereof an effective amount of the respirable dry particles or drypowder.

The invention also relates to a respirable dry powder or dry particle,as described herein, for use in therapy (e.g., treatment, prophylaxis,or diagnosis). The invention also relates to the use of a respirable dryparticle or dry powder, as described herein, for use in treatment,prevention or reducing contagion as described herein, and in themanufacture of a medicament for the treatment, prophylaxis or diagnosisof a respiratory disease and/or infection as described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1F is a table that shows properties for dry powders preparedfrom feedstock Formulations I, II, III and XIV described in Examples 1-3and 14. FIG. 1A includes spray drying parameters used for spray dryingthe powders. FIG. 1B shows the HPLC results for percent calcium ioncontent of the powders, density results including tap and bulkdensities, and Karl Fischer results for percent water content in thepowders. FIG. 1C shows fine particle fraction (FPF) data and percentmass of powders collected using a two-stage (ACI-2) Andersen CascadeImpactor. FIG. 1D shows fine particle fraction (FPF) data and percentmass of powders collected using an eight-stage (ACI-8) Andersen CascadeImpactor. FIG. 1E shows data for mass median aerodynamic diameter (MMAD)and FPF (based on total dose and recovered dose). FIG. 1F shows data forvolume median geometric diameter (DV50), geometric standard deviation(GSD) and percent volume less than 5.0 microns (V<5.0 μm) as measured bySpraytec instrument and geometric or volume particle size distribution(which is also referred to as VMGD, ×50/dg or ×50), GSD and 1/4 bar and0.5/4 bar information as measured by HELOS with RODOS attachmentinstrument.

FIG. 2 is a graph that shows a comparison between the average tap andbulk densities for particles prepared from feedstock Formulations I, IIand III and a placebo.

FIG. 3 is a graph that shows a comparison between the particles(prepared from feedstock Formulations I-III and a placebo) at differentdispersion (regulator) pressures for measured volume median geometricdiameter (×50) using a laser diffraction instrument (HELOS with RODOS).

FIG. 4 is a graph that shows a comparison between the particles preparedfrom feedstock Formulations I (identified as PUR111 (Citrate)), II(identified as PUR113 (Lactate)) and III (identified as PUR112(Sulfate)) and a placebo for average FPF obtained by an ACI-2 and ACI-8.

FIG. 5A-D are electron micrographs of Formulation I (FIG. 5A);Formulation II (FIG. 5B); Formulation III (FIG. 5C); and Formulation XIV(FIG. 5D)

FIGS. 6A-6B is a table that shows properties for dry powders prepared byfeedstock Formulations 6.1-6.9. Formulation 6.1 in FIG. 5 corresponds toFormulation II-B in Example 2. Formulation 6.4 in FIG. 5 corresponds toFormulation I-B in Example 1. Formulation 6.7 in FIG. 5 corresponds toFormulation III-B in Example 3. Abbreviations in the table heading aredescribed elsewhere in the specification. In FIG. 5, all powders weremade using a Man spray dryer.

FIG. 7 is a schematic of the pass-through model.

FIG. 8A is a graph showing the results of the bacterial pass-throughmodel with exposure to dry powders. A calcium sulfate-containing powder(4.5 ug Ca/cm² delivered dose) reduced bacterial movement through sodiumalginate mimetic. iii. FIG. 8B is a graph showing the results of thebacterial pass-through model with exposure to dry powders. The calciumsalt dry powders, prepared from the feedstock formulations A-E, testedcontained 0 ug, 4.3 ug, 6.4 ug or 10 ug of calcium. Calcium sulfate (4.3ug Ca/cm² delivered dose), calcium acetate (10 ug Ca/cm² delivered dose)and calcium lactate (6.4 ug Ca/cm² delivered dose) containing powdersreduced bacterial movement through sodium alginate mimetic.

FIG. 9 is a graph that shows the effect of the respirable dry powders,prepared from feedstock formulations 10-1 to 10-4 in Example 10A, onInfluenza A/WSN/33 (H1N1) infection in a dose-dependent manner.

FIG. 10 is a graph that shows the effect of the respirable dry powdersprepared for Example 10B on Influenza A/Panama/99/2007 (H3N2) infectionin a dose-dependent manner.

FIGS. 11A-D are graphs showing that dry powder formulations comprised ofcalcium salts and sodium chloride reduce the severity of influenza inferrets. FIG. 11A shows the changes in body temperature of ferretstreated with a calcium citrate powder compared to the control animals.FIG. 11B shows the changes in body temperature of ferrets treated with acalcium sulfate powder compared to the control animals. FIG. 11C showsthe changes in body temperature of ferrets treated with a calciumlactate powder compared to the control animals. FIG. 11D shows thechange in body temperature from baseline for each animal using areaunder the curve for the duration of the study (d0-d10). Data depict themean±SEM for each group (p=0.09 for the leucine control and lactategroup by Student t-test).

FIG. 12 is a graph showing dry powder formulations consisting ofdifferent excipients (mannitol, maltodextrin) with calcium lactate andsodium chloride reduced influenza titer at higher concentrations thanthe Formulation III powder alone.

FIGS. 13A-C are graphs showing calcium dry powder formulations vary inefficacy against different viral pathogens. Calu-3 cells exposed to noformulation were used as a control and compared to Calu-3 cells exposedto Formulation I, Formulation II, and Formulation III. The concentrationof virus released by cells exposed to each aerosol formulation wasquantified. Symbols represent the mean and standard deviation ofduplicate wells for each test.

FIG. 14 is a graph showing the emitted dose of Formulation III powder atthree different capsule fill weights (25 mg, 60 mg, 75 mg) at varyinginhalation energies.

FIG. 15 is a graph showing the particle size distribution of calciumlactate (Formulation II) powders emitted from different inhalers,characterized by the volume median diameter (Dv50) and plotted againstthe inhalation energy applied. Consistent values of Dv50 at decreasingenergy values indicate that the powder is well dispersed sinceadditional energy does not result in additional deagglomeration of theemitted powder.

FIG. 16 shows a high resolution XRPD pattern of Formulation I powder.This pattern shows that Formulation I powder consists of a combinationof crystalline sodium chloride and a poorly crystalline or amorphouscalcium citrate and potentially calcium chloride-rich phase.

FIG. 17 shows a comparison of XRPD patterns for Formulation I powderwith crystalline reflections from NaCl.

FIG. 18 shows an overlay of temperature cycling DSC thermogram ofFormulation I. A glass transition temperature of approximately 167° C.was observed via cyclic DSC for the amorphous calcium-rich phase.

FIG. 19 shows a high resolution XRPD pattern of Formulation III powder.This pattern shows that Formulation II powder consists of a combinationof crystalline sodium chloride and a poorly crystalline or amorphouscalcium lactate and potentially calcium chloride-rich phase.

FIG. 20 shows a comparison of XRPD patterns for Formulation III powderwith crystalline reflection from NaCl.

FIG. 21 shows an overlay of temperature cycling DSC thermogram ofFormulation III. A glass transition temperature of approximately 144° C.was observed via cyclic DSC for the amorphous calcium-rich phase.

FIG. 22 shows a high resolution XRPD pattern of Formulation XIV powder.

FIG. 23 shows a comparison of XRPD patterns for Formulation XIV powderwith crystalline reflection from NaCl.

FIG. 24 shows an overlay of temperature cycling DSC thermogram ofFormulation XIV. A glass transition temperature of approximately 134° C.was observed via cyclic DSC for the amorphous calcium-rich phase.

FIG. 25A shows a high resolution XRPD pattern of Formulation III powder.This pattern shows that Formulation III has some degree of crystallinecalcium salt content (calcium sulfate) present, in addition tocrystalline sodium chloride. FIG. 25B shows a comparison of XRPDpatterns for Formulation III powder with crystalline reflection fromNaCl.

FIG. 26 shows an overlay of temperature cycling DSC thermogram ofFormulation III. A glass transition temperature of approximately 159° C.was observed via cyclic DSC for the amorphous calcium-rich phase.

FIGS. 27A-H are RAMAN spectra. FIG. 27A shows RAMAN spectra for sixparticles from the Formulation I sample, and are shown overlaid. FIG.27B shows spectrum 389575-6 is background subtracted and overlaid withthe Raman spectra of calcium citrate tetrahydrate, sodium citrate, andleucine. FIG. 27C shows RAMAN spectra for eight particles from theFormulation III sample, and are shown overlaid. FIG. 27D shows spectrum388369-4 is background subtracted and overlaid with Raman spectra ofcalcium sulfate, calcium sulfate dihydrate, sodium sulfate anhydrous,and leucine. FIG. 27E shows RAMAN spectra for twelve particles from theFormulation II sample, and are shown overlaid. FIG. 27F shows spectra389576-7 and 389576-12 are background subtracted and overlaid with theRaman spectra of calcium lactate pentahydrate, and leucine. FIG. 27Gshows RAMAN spectra for twelve particles from the Formulation XIVsample, and are shown overlaid. FIG. 27H, spectrum 389577-9 isbackground subtracted and overlaid with the Raman spectra of calciumlactate pentahydrate.

FIG. 28 is a graph showing volume particle size results for FormulationIII (calcium sulfate) spray dried powders prepared from pre-mixed andstatic mixed liquid feed stocks with increasing solids concentrations.Particle size distribution broadens (increasing GSD) and median volumeparticle size significantly increases (×50) with increasing solidsconcentration in pre-mixed feed stocks. Particle size distributionremains constant with increasing solids concentration in static mixedfeed stocks, while the median volume particle size increases slightly,as expected with increasing solids concentrations.

FIG. 29 is a graph showing volume particle size distribution results forFormulation III (calcium sulfate) spray dried powders prepared frompre-mixed and static mixed liquid feed stocks with increasing solidsconcentrations. Particle size distribution broadens with increasingsolids concentration in pre-mixed feed stocks and remains narrow withincreasing solids concentration in static mixed feed stocks. Triangles 5g/L, static mixed; squares, 5 g/L, pre-mixed; diamonds, 30 g/L, staticmixed; circles 30 g/L, pre-mixed.

FIG. 30 is a graph showing aerosol characterization results forFormulation III (calcium sulfate) spray dried powders prepared frompre-mixed and static mixed liquid feed stocks with increasing solidsconcentration.

FIG. 31A-B are graphs showing the change in fine particle fraction (FPF)of formulations Formulation I (calcium citrate), Formulation II (calciumlactate) and Formulation III (calcium sulfate) during in-use stabilitytesting at extreme conditions. The graph compares change in FPF (totaldose)<5.6 microns (%) versus time elapsed in the chamber at extremetemperature and humidity conditions (30° C., 75% RH). The values in thelegend indicate the true value at time zero. The plots show fluctuationas a function of change as compared to time zero. FIG. 31B is a graphshowing change in volume particle size of formulations Formulation I(calcium citrate), Formulation II (calcium lactate) and Formulation III(calcium sulfate) during in-use stability testing at extreme conditions.The graph compares change in median volume particle size versus timeelapsed in the chamber at extreme temperature and humidity conditions(30° C., 75% RH). The values in the legend indicate the true value attime zero. The plots show fluctuation as a function of change ascompared to time zero. FIG. 31C,D show similar data for a second set ofspray-dried formulations comprised of a control calcium chloride:sodiumchloride:leucine powder and calcium lactate:sodium chloride powderscontaining 10% (i) lactose, (ii) mannitol) or (iii) maltodextrin asexcipients. FIG. 31C compares changes in FPF (total dose) <5.6 microns(%) versus time elapsed in the chamber for the second set of powders atextreme temperature and humidity conditions (30° C., 75% RH). The valuesin the legend indicate the true value at time zero. The plots showfluctuation as a function of change as compared to time zero. FIG. 31Dis a graph showing changes in volume particle sizes of the second set ofpowders during in-use stability testing at extreme conditions. The graphcompares change in median volume particle size versus time elapsed inthe chamber at extreme temperature and humidity conditions (30° C., 75%RH). The values in the legend indicate the true value at time zero. Theplots show fluctuation as a function of change as compared to time zero.

FIG. 32 is a graph showing powder stability for a range of differentpowders as measured by volume particle size upon exposure to ˜40% RHconditions for up to one week.

FIG. 33 is a graph showing volume particle size upon exposure to ˜40% RHconditions for a range of different powders for up to one week. Thisfigure is identical to FIG. 32, except that chloride was removed toallow for better detail.

FIG. 34 is a graph showing a representative TGA thermogram forFormulation I.

FIG. 35 is a graph showing heats of solution obtained upon dissolutionof FormulationS I through III. FormulationS I through III resulted insignificantly decreased heats of solution as compared to both rawcalcium chloride dihydratedihydrate and a control calciumchloride:sodium chloride:leucine powder.

FIG. 36 is a graph showing the results of an in vivo pneumonia study.Animals treated with Formulation III (calcium sulfate) exhibited 5-foldlower bacterial titers, animals treated with Formulation I (calciumcitrate) exhibited 10.4-fold lower bacterial titers, and animals treatedwith Formulation II (calcium lactate) exhibited 5.9-fold lower bacterialtiters.

FIG. 37 is a table showing the compositions of exemplary dry powderformulations.

DETAILED DESCRIPTION OF THE INVENTION

This invention relates, in part, to respirable dry powders that deliverone or more divalent metal cations, such as calcium, as an activeingredient, and to divalent metal cation-containing (e.g.,calcium-containing) respirable dry particles contained within thepowders. The invention also relates to respirable dry particles thatcontain one or more monovalent cations (such as Na⁺) and to dry powdersthat contain the respirable particles.

In one aspect, the respirable dry powders and dry particles of theinvention may be divalent metal cation (e.g., calcium) dense respirableparticles that are small and dispersible. In another aspect, therespirable dry particles may be large or small, e.g., the dry powder hasa geometric diameter (VMGD) between 0.5 microns and 30 microns.Optionally, the MMAD of the dry powder may be between 0.5 and 10microns, more preferably between 1 and 5 microns.

Respirable dry powders that contain small particles and that aredispersible in air, and preferably dense (e.g., dense in activeingredient) are a departure from the conventional wisdom. It is wellknown that the propensity for particles to aggregate or agglomerateincreases as particle size decreases. See, e.g., Hickey, A. et al.,“Factors Influencing the Dispersion of Dry Powders as Aerosols”,Pharmaceutical Technology, August, 1994.

As described herein, the invention provides respirable dry powders thatcontain respirable particles that are small and dispersible in airwithout additional energy sources beyond the subject's inhalation. Thus,the respirable dry powders and respirable dry particles can be usedtherapeutically, without including large amounts of non-activecomponents (e.g., excipients) in the particles or powders, or by usingdevices that apply mechanical forces to disrupt aggregated oragglomerated particles during or just prior to administration.

The respirable dry powders and respirable particles of the invention arealso generally, dense in active ingredient(s), i.e., divalent metalcations (e.g., calcium containing salt(s)). For example, as describedherein, when an excipient is included in the respirable dry powder orparticles, the excipient is a minor component (e.g., about 50% or less,by weight, preferably about 20% or less by weight, about 12% or less byweight, about 10% or less by weight, about 8% or less by weight or lessby weight). Thus, in one aspect, the respirable particles are not onlysmall and highly dispersible, but can contain a large amount of divalentmetal cation, for example, calcium (Ca²⁺). Accordingly, a smaller amountof powder will need to be administered in order to deliver the desireddose of divalent metal cation (e.g., calcium). For example, the desireddose of calcium may be delivered with one or two inhalations from acapsule-type or blister-type inhaler.

DEFINITIONS

The term “dry powder” as used herein refers to a composition containsfinely dispersed respirable dry particles that are capable of beingdispersed in an inhalation device and subsequently inhaled by a subject.Such dry powder or dry particle may contain up to about 15% water orother solvent, or be substantially free of water or other solvent, or beanhydrous.

The term “dry particles” as used herein refers to respirable particlesthat may contain up to about 15% water or other solvent, or besubstantially free of water or other solvent, or be anhydrous.

The term “respirable” as used herein refers to dry particles or drypowders that are suitable for delivery to the respiratory tract (e.g.,pulmonary delivery) in a subject by inhalation. Respirable dry powdersor dry particles have a mass median aerodynamic diameter (MMAD) of lessthan about 10 microns, preferably about 5 microns or less.

As used herein, the terms “administration” or “administering” ofrespirable dry particles refers to introducing respirable dry particlesto the respiratory tract of a subject.

As used herein, the term “respiratory tract” includes the upperrespiratory tract (e.g., nasal passages, nasal cavity, throat, pharynx),respiratory airways (e.g., larynx, tranchea, bronchi, bronchioles) andlungs (e.g., respiratory bronchioles, alveolar ducts, alveolar sacs,alveoli).

The term “dispersible” is a term of art that describes thecharacteristic of a dry powder or dry particles to be dispelled into arespirable aerosol. Dispersibility of a dry powder or dry particles isexpressed herein as the quotient of the volume median geometric diameter(VMGD) measured at a dispersion (i.e., regulator) pressure of 1 bardivided by the VMGD measured at a dispersion (i.e., regulator) pressureof 4 bar, or VMGD at 0.5 bar divided by the VMGD at 4 bar as measured byHELOS/RODOS. These quotients are referred to herein as “1/4 bar,” and“0.5/4 bar,” respectively, and dispersibility correlates with a lowquotient. For example, 1/4 bar refers to the VMGD of respirable dryparticles or powders emitted from the orifice of a RODOS dry powderdisperser (or equivalent technique) at about 1 bar, as measured by aHELOS or other laser diffraction system, divided the VMGD of the samerespirable dry particles or powders measured at 4 bar by HELOS/RODOS.Thus, a highly dispersible dry powder or dry particles will have a 1/4bar or 0.5/4 bar ratio that is close to 1.0. Highly dispersible powdershave a low tendency to agglomerate, aggregate or clump together and/or,if agglomerated, aggregated or clumped together, are easily dispersed orde-agglomerated as they emit from an inhaler and are breathed in by thesubject. Dispersibility can also be assessed by measuring the sizeemitted from an inhaler as a function of flowrate.

The terms “FPF (<5.6),” “FPF (<5.6 microns),” and “fine particlefraction of less than 5.6 microns” as used herein, refer to the fractionof a sample of dry particles that have an aerodynamic diameter of lessthan 5.6 microns. For example, FPF (<5.6) can be determined by dividingthe mass of respirable dry particles deposited on the stage one and onthe collection filter of a two-stage collapsed Andersen Cascade Impactor(ACI) by the mass of respirable dry particles weighed into a capsule fordelivery to the instrument. This parameter may also be identified as“FPF_TD (<5.6),” where TD means total dose. A similar measurement can beconducted using an eight-stage ACI. The eight-stage ACI cutoffs aredifferent at the standard 60 L/min flowrate, but the FPF_TD (<5.6) canbe extrapolated from the eight-stage complete data set. The eight-stageACI result can also be calculated by the USP method of using the dosecollected in the ACI instead of what was in the capsule to determineFPF.

The terms “FPF (<3.4),” “FPF (<3.4 microns),” and “fine particlefraction of less than 3.4 microns” as used herein, refer to the fractionof a mass of respirable dry particles that have an aerodynamic diameterof less than 3.4 microns. For example, FPF (<3.4) can be determined bydividing the mass of respirable dry particles deposited on thecollection filter of a two-stage collapsed ACI by the total mass ofrespirable dry particles weighed into a capsule for delivery to theinstrument. This parameter may also be identified as “FPF_TD (<3.4),”where TD means total dose. A similar measurement can be conducted usingan eight-stage ACI. The eight-stage ACI result can also be calculated bythe USP method of using the dose collected in the ACI instead of whatwas in the capsule to determine FPF.

The terms “FPF (<5.0),” “FPF (<5.0 microns),” and “fine particlefraction of less than 5.0 microns” as used herein, refer to the fractionof a mass of respirable dry particles that have an aerodynamic diameterof less than 5.0 microns. For example, FPF (<5.0) can be determined byusing an eight-stage ACI at the standard 60 L/min flowrate byextrapolating from the eight-stage complete data set. This parameter mayalso be identified as “FPF_TD (<5.0),” where TD means total dose.

As used herein, the term “emitted dose” or “ED” refers to an indicationof the delivery of a drug formulation from a suitable inhaler deviceafter a firing or dispersion event. More specifically, for dry powderformulations, the ED is a measure of the percentage of powder that isdrawn out of a unit dose package and that exits the mouthpiece of aninhaler device. The ED is defined as the ratio of the dose delivered byan inhaler device to the nominal dose (i.e., the mass of powder per unitdose placed into a suitable inhaler device prior to firing). The ED isan experimentally-measured parameter, and can be determined using themethod of USP Section 601 Aerosols, Metered-Dose Inhalers and Dry PowderInhalers, Delivered-Dose Uniformity, Sampling the Delivered Dose fromDry Powder Inhalers, United States Pharmacopia convention, Rockville,Md., 13^(th) Revision, 222-225, 2007. This method utilizes an in vitrodevice set up to mimic patient dosing.

The term “effective amount,” as used herein, refers to the amount ofagent needed to achieve the desired effect, such as an amount that issufficient to increase surface and/or bulk viscoelasticy of therespiratory tract mucus (e.g., airway lining fluid), increase gelationof the respiratory tract mucus (e.g., at the surface and/or bulkgelation), increase surface tension of the respiratory tract mucus,increasing elasticity of the respiratory tract mucus (e.g., surfaceelasticity and/or bulk elasticity), increase surface viscosity of therespiratory tract mucus (e.g., surface viscosity and/or bulk viscosity),reduce the amount of exhaled particles, reduce pathogen (e.g., bacteria,virus) burden, reduce symptoms (e.g., fever, coughing, sneezing, nasaldischarge, diarrhea and the like), reduce occurrence of infection,reduce viral replication, or improve or prevent deterioration ofrespiratory function (e.g., improve forced expiratory volume in 1 secondFEV1 and/or forced expiratory volume in 1 second FEV1 as a proportion offorced vital capacity FEV1/FVC, reduce bronchoconstriction). The actualeffective amount for a particular use can vary according to theparticular dry powder or dry particle, the mode of administration, andthe age, weight, general health of the subject, and severity of thesymptoms or condition being treated. Suitable amounts of dry powders anddry particles to be administered, and dosage schedules, for a particularpatient can be determined by a clinician of ordinary skill based onthese and other considerations.

The term “pharmaceutically acceptable excipient” as used herein meansthat the excipient can be taken into the lungs with no significantadverse toxicological effects on the lungs. Such excipient are generallyregarded as safe (GRAS) by the U.S. Food and Drug Administration.

Dry Powders and Dry Particles

The invention relates to respirable dry powders and dry particles thatcontain one or more divalent metal cations, such as beryllium (Be²⁺),magnesium, (Mg²⁺), calcium (Ca²⁺), strontium (Sr²⁺), barium (Ba²⁺),radium (Ra²⁺), or iron (ferrous ion, Fe²⁺), as an active ingredient. Theactive divalent metal cation (e.g., calcium) is generally present in thedry powders and dry particles in the form of a salt, which can becrystalline or amorphous. The dry powders and dry particles canoptionally include additional salts (e.g. monovalent salts, such assodium salts, potassium salts, and lithium salts.), therapeuticallyactive agents or pharmaceutically acceptable excipients.

In some aspects, the respirable dry powder and dry particles contain oneor more salts of a group IIA element (i.e., one or more beryllium salts,magnesium salts, calcium salts, barium salts, radium salts or anycombination of the forgoing). In more particular aspects, the respirabledry powder and dry particles contain one or more calcium salts,magnesium salts or any combination of the forgoing. In particularembodiments, the respirable dry powder and dry particles contain one ormore calcium salts. In other particular embodiments, respirable drypowder and dry particles contain one or more magnesium salts.

Suitable beryllium salts include, for example, beryllium phosphate,beryllium acetate, beryllium tartrate, beryllium citrate, berylliumgluconate, beryllium maleate, beryllium succinate, sodium berylliummalate, beryllium alpha brom camphor sulfonate, berylliumacetylacetonate, beryllium formate or any combination thereof.

Suitable magnesium salts include, for example, magnesium fluoride,magnesium chloride, magnesium bromide, magnesium iodide, magnesiumphosphate, magnesium sulfate, magnesium sulfite, magnesium carbonate,magnesium oxide, magnesium nitrate, magnesium borate, magnesium acetate,magnesium citrate, magnesium gluconate, magnesium maleate, magnesiumsuccinate, magnesium malate, magnesium taurate, magnesium orotate,magnesium glycinate, magnesium naphthenate, magnesium acetylacetonate,magnesium formate, magnesium hydroxide, magnesium stearate, magnesiumhexafluorosilicate, magnesium salicylate or any combination thereof.

Suitable calcium salts include, for example, calcium chloride, calciumsulfate, calcium lactate, calcium citrate, calcium carbonate, calciumacetate, calcium phosphate, calcium alginite, calcium stearate, calciumsorbate, calcium gluconate and the like.

Suitable strontium salts include, for example, strontium chloride,strontium phosphate, strontium sulfate, strontium carbonate, strontiumoxide, strontium nitrate, strontium acetate, strontium tartrate,strontium citrate, strontium gluconate, strontium maleate, strontiumsuccinate, strontium malate, strontium aspartate in either L and/orD-form, strontium fumarate, strontium glutamate in either L- and/orD-form, strontium glutarate, strontium lactate, strontium L-threonate,strontium malonate, strontium ranelate (organic metal chelate),strontium ascorbate, strontium butyrate, strontium clodronate, strontiumibandronate, strontium salicylate, strontium acetyl salicylate or anycombination thereof.

Suitable barium salts include, for example, barium hydroxide, bariumfluoride, barium chloride, barium bromide, barium iodide, bariumsulfate, barium sulfide (S), barium carbonate, barium peroxide, bariumoxide, barium nitrate, barium acetate, barium tartrate, barium citrate,barium gluconate, barium maleate, barium succinate, barium malate,barium glutamate, barium oxalate, barium malonate, barium naphthenate,barium acetylacetonate, barium formate, barium benzoate, bariump-t-butylbenzoate, barium adipate, barium pimelate, barium suberate,barium azelate, barium sebacate, barium phthalate, barium isophthalate,barium terephthalate, barium anthranilate, barium mandelate, bariumsalicylate, barium titanate or any combination thereof.

Suitable radium salts included, for example, radium fluoride, radiumchloride, radium bromide, radium iodide, radium oxide, radium nitride orany combination thereof.

Suitable iron (ferrous) salts include, for example, ferrous sulfate,ferrous oxides, ferrous acetate, ferrous citrate, ferrous ammoniumcitrate, ferrous ferrous gluconate, ferrous oxalate, ferrous fumarate,ferrous maleate, ferrous malate, ferrous lactate, ferrous ascorbate,ferrous erythrobate, ferrous glycerate, ferrous pyruvate or anycombination thereof.

In one aspect, the dry particles of the invention are small, andpreferably divalent metal cation (e.g., calcium) dense, and aredispersible. The size of the dry particles can be expressed in a varietyof ways that are conventional in the art, such as, fine particlefraction (FPF), volumetric median geometric diameter (VMGD), or massmedian aerodynamic diameter (MMAD). Generally, the dry particles of theinvention have a VMGD as measured by HELOS/RODOS at 1.0 bar of about 10μm or less (e.g., about 0.1 μm to about 10 μm). Preferably, the dryparticles of the invention have an VMGD of about 9 μm or less (e.g.,about 0.1 μm to about 9 μm), about 8 μm or less (e.g., about 0.1 μm toabout 8 μm), about 7 μm or less (e.g., about 0.1 μm to about 7 μm),about 6 μm or less (e.g., about 0.1 μm to about 6 μm), about 5 μm orless (e.g., less than 5 μm, about 0.1 μm to about 5 μm), about 4 μm orless (e.g., 0.1 μm to about 4 μm), about 3 μm or less (e.g., 0.1 μm toabout 3 μm), about 2 μm or less (e.g., 0.1 μm to about 2 μm), about 1 μmor less (e.g., 0.1 μm to about 1 μm), about 1 μm to about 6 μm, about 1μm to about 5 μm, about 1 μm to about 4 μm, about 1 μm to about 3 μm, orabout 1 μm to about 2 μm as measured by HELOS/RODOS at 1.0 bar.

In another aspect, the dry particles of the invention are large, andpreferably calcium dense, and are dispersible. Generally, the dryparticles of the invention have a VMGD as measured by HELOS/RODOS at 1.0bar of about 30 μm or less (e.g., about 5 μm to about 30 μm).Preferably, the dry particles of the invention have an VMGD of about 25μm or less (e.g., about 5 μm to about 25 μm), about 20 μm or less (e.g.,about 5 μm to about 20 μm), about 15 μm or less (e.g., about 5 μm toabout 15 μm), about 12 μm or less (e.g., about 5 μm to about 12 μm),about 10 μm or less (e.g., about 5 μm to about 10 μm), or about 8 μm orless (e.g., 6 μm to about 8 μm) as measured by HELOS/RODOS at 1.0 bar.

In addition, whether the particles are small or large, the dry particlesof the invention are dispersible, and have 1/4 bar and/or 0.5/4 bar ofabout 2.2 or less (e.g., about 1.0 to about 2.2) or about 2.0 or less(e.g., about 1.0 to about 2.0). Preferably, the dry particles of theinvention have 1/4 bar and/or 0.5/4 bar of about 1.9 or less (e.g.,about 1.0 to about 1.9), about 1.8 or less (e.g., about 1.0 to about1.8), about 1.7 or less (e.g., about 1.0 to about 1.7), about 1.6 orless (e.g., about 1.0 to about 1.6), about 1.5 or less (e.g., about 1.0to about 1.5), about 1.4 or less (e.g., about 1.0 to about 1.4), about1.3 or less (e.g., less than 1.3, about 1.0 to about 1.3), about 1.2 orless (e.g., 1.0 to about 1.2), about 1.1 or less (e.g., 1.0 to about 1.1μm) or the dry particles of the invention have 1/4 bar of about 1.0.

Alternatively or in addition, the respirable dry particles of theinvention can have an MMAD of about 10 microns or less, such as an MMADof about 0.5 micron to about 10 microns. Preferably, the dry particlesof the invention have an MMAD of about 5 microns or less (e.g. about 0.5micron to about 5 microns, preferably about 1 micron to about 5microns), about 4 microns or less (e.g., about 1 micron to about 4microns), about 3.8 microns or less (e.g. about 1 micron to about 3.8microns), about 3.5 microns or less (e.g. about 1 micron to about 3.5microns), about 3.2 microns or less (e.g. about 1 micron to about 3.2microns), about 3 microns or less (e.g. about 1 micron to about 3.0microns), about 2.8 microns or less (e.g. about 1 micron to about 2.8microns), about 2.2 microns or less (e.g. about 1 micron to about 2.2microns), about 2.0 microns or less (e.g. about 1 micron to about 2.0microns) or about 1.8 microns or less (e.g. about 1 micron to about 1.8microns).

Alternatively or in addition, the respirable dry powders and dryparticles of the invention can have an FPF of less than about 5.6microns (FPF<5.6 μm) of at least about 20%, at least about 30%, at leastabout 40%, preferably at least about 45%, at least about 50%, at leastabout 55%, at least about 60%, at least about 65%, or at least about70%.

Alternatively or in addition, the dry powders and dry particles of theinvention have a FPF of less than 5.0 microns (FPF_TD<5.0 μm) of atleast about 20%, at least about 30%, at least about 45%, preferably atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 65% or at least about 70%. Alternatively or inaddition, the dry powders and dry particles of the invention have a FPFof less than 5.0 microns of the emitted dose (FPF_ED<5.0 μm) of at leastabout 45%, preferably at least about 50%, at least about 60%, at leastabout 65%, at least about 70%, at least about 75%, at least about 80%,or at least about 85%. Alternatively or in addition, the dry powders anddry particles of the invention can have an FPF of less than about 3.4microns (FPF<3.4 μm) of at least about 20%, preferably at least about25%, at least about 30%, at least about 35%, at least about 40%, atleast about 45%, at least about 50%, or at least about 55%.

Alternatively or in addition, the respirable dry powders and dryparticles of the invention have a tap density of about 0.1 g/cm³ toabout 1.0 g/cm³. For example, the small and dispersible dry particleshave a tap density of about 0.1 g/cm³ to about 0.9 g/cm³, about 0.2g/cm³ to about 0.9 g/cm³, about 0.2 g/cm³ to about 0.9 g/cm³, about 0.3g/cm³ to about 0.9 g/cm³, about 0.4 g/cm³ to about 0.9 g/cm³, about 0.5g/cm³ to about 0.9 g/cm³, or about 0.5 g/cm³ to about 0.8 g/cm³, greaterthan about 0.4 g/cc, greater than about 0.5 g/cc, greater than about 0.6g/cc, greater than about 0.7 g/cc, about 0.1 g/cm³ to about 0.8 g/cm³,about 0.1 g/cm³ to about 0.7 g/cm³, about 0.1 g/cm³ to about 0.6 g/cm³,about 0.1 g/cm³ to about 0.5 g/cm³, about 0.1 g/cm³ to about 0.4 g/cm³,about 0.1 g/cm³ to about 0.3 g/cm³, less than 0.3 g/cm³. In a preferredembodiment, tap density is greater than about 0.4 g/cc. In anotherpreferred embodiment, tap density is greater than about 0.5 g/cc.Alternatively, tap density is less than about 0.4 g/cc.

Alternatively or in addition, the respirable dry powders and dryparticles of the invention can have a water or solvent content of lessthan about 15% by weight of the respirable dry particle. For example,the respirable dry particles of the invention can have a water orsolvent content of less than about 15% by weight, less than about 13% byweight, less than about 11.5% by weight, less than about 10% by weight,less than about 9% by weight, less than about 8% by weight, less thanabout 7% by weight, less than about 6% by weight, less than about 5% byweight, less than about 4% by weight, less than about 3% by weight, lessthan about 2% by weight, less than about 1% by weight or be anhydrous.The respirable dry particles of the invention can have a water orsolvent content of less than about 6% and greater than about 1%, lessthan about 5.5% and greater than about 1.5%, less than about 5% andgreater than about 2%, about 2%, about 2.5%, about 3%, about 3.5%, about4%, about 4.5% about 5%.

As described herein, the respirable dry particles of the inventioncontain one or more divalent metal cations (e.g., calcium (Ca²⁺)) as anactive ingredient which is generally present in the form of a salt(e.g., crystalline and/or amorphous). Suitable calcium salts that can bepresent in the respirable dry particles of the invention include, forexample, calcium chloride, calcium sulfate, calcium lactate, calciumcitrate, calcium carbonate, calcium acetate, calcium phosphate, calciumalginite, calcium stearate, calcium sorbate, calcium gluconate and thelike. In certain preferred aspects, the dry powder or dry particles ofthe invention do not contain calcium phosphate, calcium carbonate,calcium alginate, calcium stearate or calcium gluconate. In anotherpreferred aspect, the dry powder or dry particles of the inventioninclude calcium citrate, calcium lactate, calcium chloride, calciumsulfate, or any combination of these salts. In another preferred aspect,the dry powder or dry particles include calcium citrate, calciumlactate, or any combination of the these salts. If desired, therespirable dry particles of the invention contain a divalent metalcation salt (e.g., a calcium salt) and further contain one or moreadditional salts, such as one or more non-toxic salts of the elementssodium, potassium, magnesium, calcium, aluminum, silicon, scandium,titanium, vanadium, chromium, cobalt, nickel, copper, manganese, zinc,tin, silver and the like. Preferably, the dry particles contain at leastone calcium salt and at least one monovalent cation salt (e.g., a sodiumsalt).

Suitable sodium salts that can be present in the respirable dryparticles of the invention include, for example, sodium chloride, sodiumcitrate, sodium sulfate, sodium lactate, sodium acetate, sodiumbicarbonate, sodium carbonate, sodium stearate, sodium ascorbate, sodiumbenzoate, sodium biphosphate, sodium phosphate, sodium bisulfite, sodiumborate, sodium gluconate, sodium metasilicate and the like. In apreferred aspect, the dry powders and dry particles include sodiumchloride, sodium citrate, sodium lactate, sodium sulfate, or anycombination of these salts.

Suitable lithium salts include, for example, lithium chloride, lithiumbromide, lithium carbonate, lithium nitrate, lithium sulfate, lithiumacetate, lithium lactate, lithium citrate, lithium aspartate, lithiumgluconate, lithium malate, lithium ascorbate, lithium orotate, lithiumsuccinate or and combination thereof.

Suitable potassium salts include, for example, potassium chloride,potassium bromide, potassium iodide, potassium bicarbonate, potassiumnitrite, potassium persulfate, potassium sulfite, potassium bisulfite,potassium phosphate, potassium acetate, potassium citrate, potassiumglutamate, dipotassium guanylate, potassium gluconate, potassium malate,potassium ascorbate, potassium sorbate, potassium succinate, potassiumsodium tartrate and any combination thereof.

Preferred divalent metal salts (e.g., calcium salts) have one,preferably two or more of the following characteristics: (i) can beprocessed into a respirable dry particle, (ii) possess sufficientphysicochemical stability in dry powder form to facilitate theproduction of a powder that is dispersible and physically stable over arange of conditions, including upon exposure to elevated humidity, (iii)undergo rapid dissolution upon deposition in the lungs, for example,half of the mass of the cation of the divalent metal can dissolved inless than 30 minutes, less than 15 minutes, less than 5 minutes, lessthan 2 minutes, less than 1 minute, or less than 30 seconds, and (iv) donot possess properties that can result in poor tolerability or adverseevents, such as a significant exothermic or endothermic heat of solution(ΔH). for example, a ΔH lower than of about −10 kcal/mol or greater thanabout 10 kcal/mol. Rather, a preferred AH is between about −9 kcal/moland about 9 kcal/mol, between about −8 kcal/mol and about 8 kcal/mol,between about −7 kcal/mol and about 7 kcal/mol, between about −6kcal/mol and about 6 kcal/mol, between about −5 kcal/mol and about 5kcal/mol, between about −4 kcal/mol and about 4 kcal/mol, between about−3 kcal/mol and about 3 kcal/mol, between about −2 kcal/mol and about 2kcal/mol, between about −1 kcal/mol and about 1 kcal/mol, or about 0kcal/mol

Regarding the dissolution rate upon deposition of the dry powder orparticles in the lungs, an alternative to rapid dissolution of theparticles in the lungs, the divalent metal salt undergoes sustaineddissolution upon deposition. The period of sustained dissolution, in oneaspect, is on the time scale of minutes, for example half of the cationof the divalent metal salt can be released from the particle in greaterthan about 30 minutes or greater than about 45 minutes. In anotheraspect, the period of sustained dissolution is over a time scale ofhours, for example half of the divalent metal salt can be released ingreater than about 1 hour, greater than 1.5 hours, greater than about 2hours, greater than about 4 hours, greater than about 8 hours, orgreater than about 12 hours. In a further aspect, the sustaindissolution is over a period of one day or two days.

Suitable divalent metal cation salts (e.g., calcium salts) can havedesired solubility characteristics. In general, highly or moderatelysoluble divalent metal cation salts (e.g., calcium salts) are preferred.For example, suitable divalent metal cation salts (e.g., calcium salts)that are contained in the respirable dry particles and dry powders canhave a solubility in distilled water at room temperature (20-30° C.) and1 bar of at least about 0.4 g/L, at least about 0.85 g/L, at least about0.90 g/L, at least about 0.95 g/L, at least about 1.0 g/L, at leastabout 2.0 g/L, at least about 5.0 g/L, at least about 6.0 g/L, at leastabout 10.0 g/L, at least about 20 g/L, at least about 50 g/L, at leastabout 90 g/L, at least about 120 g/L, at least about 500 g/L, at leastabout 700 g/L or at least about 1000 g/L. Preferably, the divalent metalcation salt has a solubility greater than about 0.90 g/L, greater thanabout 2.0 g/L, or greater than about 90 g/L.

Dry particles and dry powders of the invention can be prepared, ifdesired, that contain divalent metal cation salts (e.g., calcium salts)that are not highly soluble in water. As described herein, such dryparticles and dry powders can be prepared using a feed stock of adifferent, more soluble salt, and permitting anion exchange to producethe desired divalent metal cation salts (e.g., calcium salt) prior to orconcurrently with spray drying.

Dry powder and particles of the invention may contain a high percentageof active ingredient (e.g., divalent metal cation (e.g., calcium)) inthe composition, and be divalent metal cation dense. The dry particlesmay contain 3% or more, 5% or more, 10% or more, 15% or more, 20% oremore, 25% or more, 30% or more, 35% or more, 40% or more, 50% or more,60% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% ormore, or 95% or more active ingredient.

It is advantageous when the divalent metal cation salt (e.g., calciumsalt) dissociates to provide two or more moles of divalent metal cation(e.g., Ca²⁺) per mole of salt. Such salts can be used to producerespirable dry powders and dry particles that are dense in divalentmetal cation (e.g., calcium). For example, one mole of calcium citrateprovides three moles of Ca²⁺ upon dissolution. It is also generallypreferred that the divalent metal cation salt (e.g., calcium salt) is asalt with a low molecular weight and/or contain low molecular weightanions. Low molecular weight divalent metal cation salts, such ascalcium salts that contain calcium ions and low molecular weight anions,are divalent cation dense (e.g., Ca²⁺) dense relative to high molecularsalts and salts that contain high molecular weight anions. It isgenerally preferred that the divalent metal cation salt (e.g., calciumsalt) has a molecular weight of less than about 1000 g/mol, less thanabout 950 g/mol, less than about 900 g/mol, less than about 850 g/mol,less than about 800 g/mol, less than about 750 g/mol, less than about700 g/mol, less than about 650 g/mol, less than about 600 g/mol, lessthan about 550 g/mol, less than about 510 g/mol, less than about 500g/mol, less than about 450 g/mol, less than about 400 g/mol, less thanabout 350 g/mol, less than about 300 g/mol, less than about 250 g/mol,less than about 200 g/mol, less than about 150 g/mol, less than about125 g/mol, or less than about 100 g/mol. In addition or alternatively,it is generally preferred that the divalent metal cation (e.g., calciumion) contributes a substantial portion of the weight to the overallweight of the divalent metal cation salt. It is generally preferred thatthe divalent metal cation (e.g., calcium ion) contribute at least 10% ofthe weight of the overall salt, at least 16%, at least 20%, at least24.5%, at least 26%, at least 31%, at least 35%, or at least 38% of theweight of the overall divalent metal cation salt (e.g., calcium salt).

Alternatively or in addition, the respirable dry particles of theinvention can include a suitable divalent metal cation salt (e.g.,calcium salt) that provides divalent metal cation (Ca²⁺), wherein theweight ratio of divalent metal cation (e.g., calcium ion) to the overallweight of said salt is between about 0.1 to about 0.5. For example, theweight ratio of divalent methal cation (e.g, calcium ion) to the overallweight of said salt is between about 0.15 to about 0.5, between about0.18 to about 0.5, between about 0.2 to about 5, between about 0.25 toabout 0.5, between about 0.27 to about 0.5, between about 0.3 to about5, between about 0.35 to about 0.5, between about 0.37 to about 0.5, orbetween about 0.4 to about 0.5.

Alternatively or in addition, the respirable dry particles of theinvention can contain a divalent metal cation salt (e.g., calcium salt)which provides divalent cation (e.g., Ca²⁺) in an amount of at leastabout 5% by weight of the respirable dry particles. For example, therespirable dry particles of the invention can include a divalent metalcation salt (e.g., calcium salt) which provides divalent cation (e.g.,Ca²⁺) in an amount of at least about 7% by weight, at least about 10% byweight, at least about 11% by weight, at least about 12% by weight, atleast about 13% by weight, at least about 14% by weight, at least about15% by weight, at least about 17% by weight, at least about 20% byweight, at least about 25% by weight, at least about 30% by weight, atleast about 35% by weight, at least about 40% by weight, at least about45% by weight, at least about 50% by weight, at least about 55% byweight, at least about 60% by weight, at least about 65% by weight or atleast about 70% by weight of the respirable dry particles.

Alternatively or in addition, the respirable dry particles of theinvention can contain a divalent metal cation salt which providesdivalent metal cation (e.g., Ca²⁺, Be²⁺, Mg²⁺, Sr²⁺, Ba²⁺, Fe²⁺) in anamount of at least about 5% by weight of the respirable dry particlesand also contain a monovalent salt (e.g, sodium salt, lithium salt,potassium salt) which provides monovalent cation (e.g, Na⁺, Li⁺, K⁺) inan amount of at least about 3% by weight of the respirable dryparticles. For example, the respirable dry particles of the inventioncan include a divalent metal cation salt (e.g., calcium salt) whichprovides divalent cation (e.g., Ca²⁺) in an amount of at least about 7%by weight, at least about 10% by weight, at least about 11% by weight,at least about 12% by weight, at least about 13% by weight, at leastabout 14% by weight, at least about 15% by weight, at least about 17% byweight, at least about 20% by weight, at least about 25% by weight, atleast about 30% by weight, at least about 35% by weight, at least about40% by weight, at least about 45% by weight, at least about 50% byweight, at least about 55% by weight, at least about 60% by weight, atleast about 65% by weight or at least about 70% by weight of therespirable dry particles; and further contain a monovalent salt sodiumsalt which provides monovalent anion (Na⁺) in an amount of at leastabout 3%, at least about 4%, at least about 5%, at least about 6%, atleast about 7%, at least about 8%, at least about 9%, at least about10%, at least about 11%, at least about 12%, at least about 14%, atleast about 16%, at least about 18%, at least about 20%, at least about22%, at least about 25%, at least about 27%, at least about 29%, atleast about 32%, at least about 35%, at least about 40%, at least about45%, at least about 50% or at least about 55% by weight of therespirable dry particles.

Alternatively or in addition, the respirable dry particles of theinvention contain a divalent metal cation salt and a monovalent cationsalt, where the divalent cation, as a component of one or more salts, ispresent in an amount of at least 5% by weight of dry particle, and theweight ratio of divalent cation to monovalent cation is about 50:1(i.e., about 50 to about 1) to about 0.1:1 (i.e., about 0.1 to about 1).The weight ratio of divalent metal cation to monovalent cation, is basedon the amount of divalent metal cation and monovalent cation that arecontained in the divalent metal cation salt and monovalent salts,respectively, that are contained in the dry particle. In particularexamples, the weight ratio of divalent metal cation to monovalent cationis about 0.2:1, about 0.3:1, about 0.4:1, about 0.5:1, about 0.6:1,about 0.7:1, about 0.8:1, about 0.86:1, about 0.92:1, about 1:1; about1.3:1, about 2:1, about 5:1, about 10:1, about 15:1, about 20:1, about25:1, about 30:1, about 35:1, about 40:1, about 45:1, or about 50:1,about 20:1 to about 0.1:1, about 15:1 to about 0.1:1, about 10:1 toabout 0.1:1, or about 5:1 to about 0.1:1.

Alternatively or in addition, the respirable dry particles of theinvention can contain a divalent metal cation salt and a monovalentcation salt, in which the divalent metal cation salt and the monovalentcation salt contain chloride, lactate, citrate or sulfate as the counterion, and the ratio of divalent metal cation (e.g., Ca²⁺, Be²⁺, Mg²⁺,Sr²⁺, Ba²⁺, Fe²⁺) to monovalent cation (e.g, Na⁺, K⁺) mole:mole is about50:1 (i.e., about 50 to about 1) to about 0.1:1 (i.e., about 0.1 toabout 1). The mole ratio of divalent metal cation to monovalent cation,is based on the amount of divalent metal cation and monovalent cationthat are contained in the divalent metal cation salt and monovalentcation salt, respectively, that are contained in the dry particle.Preferably, divalent metal cation, as a component of one or moredivalent metal cation salts, is present in an amount of at least 5% byweight of the respirable dry particle. In particular examples, divalentmetal cation and monovalent cation are present in the respirable dryparticles in a mole ratio of about 8.0:1, about 7.5:1, about 7.0:1,about 6.5:1, about 6.0:1, about 5.5:1, about 5.0:1, about 4.5:1, about4.0:1, about 3.5:1, about 3.0:1, about 2.5:1, about 2.0:1, about 1.5:1,about 1.0:1, about 0.77:1, about 0.65:1, about 0.55:1, about 0.45:1,about 0.35:1, about 0.25:1, or about 0.2:1, about 8.0:1 to about 0.55:1,about 7.0:1 to about 0.55:1, about 6.0:1 to about 0.55:1, about 5.0:1 toabout 0.55:1, about 4.0:1 to about 0.55:1, about 3.0:1 to about 0.55:1,about 2.0:1 to about 0.55:1, or about 1.0:1 to about 0.55:1.

Preferred respirable dry particles contain at least one calcium saltselected from the group consisting of calcium lactate, calcium citrate,calcium sulfate, and calcium chloride, and also contain sodium chloride.

Calcium citrate, calcium sulfate and calcium lactate possess sufficientaqueous solubility to allow for their processing into respirable drypowders via spray-drying and to facilitate their dissolution upondeposition in the lungs, yet possess a low enough hygroscopicity toallow for the production of dry powders with high calcium salt loadsthat are relatively physically stable upon exposure to normal andelevated humidity. Calcium citrate, calcium sulfate and calcium lactatealso have a significantly lower heat of solution than calcium chloride,which is beneficial for administration to the respiratory tract, andcitrate, sulfate and lactate ions are safe and acceptable for inclusionin pharmaceutical compositions.

Accordingly, in addition to any combination of the features andproperties described herein, the respirable dry particles of theinvention can contain one or more salts in a total amount of at leastabout 51% by weight of the respirable dry particles; wherein each of theone or more salts independently consists of a cation selected from thegroup consisting of calcium and sodium and an anion selected from thegroup consisting of lactate (C₃H₅O₃ ⁻), chloride (Cl⁻) citrate (C₆H₅O₇³⁻) and sulfate (SO₄ ²⁻), with the proviso that at least one of thesalts is a calcium salt. For example, the respirable dry particles ofthe invention can include one or more of the salts in a total amount ofat least about 55%, at least about 60%, at least about 65%, at leastabout 70%, at least about 75%, at least about 80%, at least about 85%,at least about 90%, at least about 91%, at least about 92%, or at leastabout 95% by weight of the respirable dry particles.

Alternatively or in addition, the respirable dry particles of theinvention can contain a calcium salt and a sodium salt, where thecalcium cation, as a component of one or more calcium salts, is presentin an amount of at least 5% by weight of the dry particle, and theweight ratio of calcium ion to sodium ion is about 50:1 (i.e., about 50to about 1) to about 0.1:1 (i.e., about 0.1 to about 1). The weightratio of calcium ion to sodium ion, is based on the amount of calciumion and sodium ion that are contained in the calcium salt and sodiumsalts, respectively, that are contained in the dry particle. Inparticular examples, the weight ratio of calcium ion to sodium ion isabout 0.2:1, about 0.3:1, about 0.4:1, about 0.5:1, about 0.6:1, about0.7:1, about 0.8:1, about 0.86:1, about 0.92:1, about 1:1; about 1.3:1,about 2:1, about 5:1, about 10:1, about 15:1, about 20:1, about 25:1,about 30:1, about 35:1, about 40:1, about 45:1, or about 50:1, about20:1 to about 0.1:1, about 15:1 to about 0.1:1, about 10:1 to about0.1:1, or about 5:1 to about 0.1:1.

Alternatively or in addition, the respirable dry particles of theinvention can contain a calcium salt and a sodium salt, in which thecalcium salt and the sodium salt contain chloride, lactate, citrate orsulfate as the counter ion, and the ratio of calcium to sodium mole:moleis about 50:1 (i.e., about 50 to about 1) to about 0.1:1 (i.e., about0.1 to about 1). The mole ratio of calcium to sodium, is based on theamount of calcium and sodium that are contained in the calcium salt andsodium salt, respectively, that are contained in the dry particle.Preferably, calcium, as a component of one or more calcium salts, ispresent in an amount of at least 5% by weight of the respirable dryparticle. In particular examples, calcium and sodium are present in therespirable dry particles in a mole ratio of about 8.0:1, about 7.5:1,about 7.0:1, about 6.5:1, about 6.0:1, about 5.5:1, about 5.0:1, about4.5:1, about 4.0:1, about 3.5:1, about 3.0:1, about 2.5:1, about 2.0:1,about 1.5:1, about 1.0:1, about 0.77:1, about 0.65:1, about 0.55:1,about 0.45:1, about 0.35:1, about 0.25:1, or about 0.2:1, about 8.0:1 toabout 0.55:1, about 7.0:1 to about 0.55:1, about 6.0:1 to about 0.55:1,about 5.0:1 to about 0.55:1, about 4.0:1 to about 0.55:1, about 3.0:1 toabout 0.55:1, about 2.0:1 to about 0.55:1, or about 1.0:1 to about0.55:1.

If desired, the respirable dry particles described herein can include aphysiologically or pharmaceutically acceptable carrier or excipient. Forexample, a pharmaceutically-acceptable excipient includes any of thestandard carbohydrate, sugar alcohol, and amino acid carriers that areknown in the art to be useful excipients for inhalation therapy, eitheralone or in any desired combination. These excipients are generallyrelatively free-flowing particulates, do not thicken or polymerize uponcontact with water, are toxicologically innocuous when inhaled as adispersed powder and do not significantly interact with the active agentin a manner that adversely affects the desired physiological action ofthe salts of the invention. Carbohydrate excipients that are useful inthis regard include the mono- and polysaccharides. Representativemonosaccharides include carbohydrate excipients such as dextrose(anhydrous and the monohydrate; also referred to as glucose and glucosemonohydrate), galactose, mannitol, D-mannose, sorbose and the like.Representative disaccharides include lactose, maltose, sucrose,trehalose and the like. Representative trisaccharides include raffinoseand the like. Other carbohydrate excipients include maltodextrin andcyclodextrins, such as 2-hydroxypropyl-beta-cyclodextrin can be used asdesired. Representative sugar alcohols include mannitol, sorbitol andthe like.

Suitable amino acid excipients include any of the naturally occurringamino acids that form a powder under standard pharmaceutical processingtechniques and include the non-polar (hydrophobic) amino acids and polar(uncharged, positively charged and negatively charged) amino acids, suchamino acids are of pharmaceutical grade and are generally regarded assafe (GRAS) by the U.S. Food and Drug Administration. Representativeexamples of non-polar amino acids include alanine, isoleucine, leucine,methionine, phenylalanine, proline, tryptophan and valine.Representative examples of polar, uncharged amino acids include cystine,glycine, glutamine, serine, threonine, and tyrosine. Representativeexamples of polar, positively charged amino acids include arginine,histidine and lysine. Representative examples of negatively chargedamino acids include aspartic acid and glutamic acid. These amino acidsare generally available from commercial sources that providepharmaceutical-grade products such as the Aldrich Chemical Company,Inc., Milwaukee, Wis. or Sigma Chemical Company, St. Louis, Mo.

Preferred amino acid excipients, such as the hydrophobic amino acidleucine, can be present in the dry particles of the invention in anamount of about 50% or less by weight of respirable dry particles. Forexample, the respirable dry particles of the invention can contain theamino acid leucine in an amount of about 5% to about 30% by weight,about 10% to about 20% by weight, about 5% to about 20% by weight, about45% or less by weight, about 40% or less by weight, about 35% or less byweight, about 30% or less by weight, about 25% or less by weight, about20% or less by weight, about 18% or less by weight, about 16% or less byweight, about 15% or less by weight, about 14% or less by weight, about13% or less by weight, about 12% or less by weight, about 11% or less byweight, about 10% or less by weight, about 9% or less by weight, about8% or less by weight, about 7% or less by weight, about 6% or less byweight, about 5% or less by weight, about 4% or less by weight, about 3%or less by weight, about 2% or less by weight, or about 1% or less byweight.

Preferred carbohydrate excipients, such as maltodextrin and mannitol,can be present in the dry particles of the invention in an amount ofabout 50% or less by weight of respirable dry particles. For example,the respirable dry particles of the invention can contain maltodextrinin an amount of about 45% or less by weight, about 40% or less byweight, about 35% or less by weight, about 30% or less by weight, about25% or less by weight, about 20% or less by weight, about 18% or less byweight, about 16% or less by weight, about 15% or less by weight, about14% or less by weight, about 13% or less by weight, about 12% or less byweight, about 11% or less by weight, about 10% or less by weight, about9% or less by weight, about 8% or less by weight, about 7% or less byweight, about 6% or less by weight, about 5% or less by weight, about 4%or less by weight, about 3% or less by weight, about 2% or less byweight, or about 1% or less by weight. In some preferred aspects, thedry particles contain an excipient selected from leucine, maltodextrin,mannitol and any combination thereof. In particular embodiments, theexcipient is leucine, maltodextrin, or mannitol.

In particular embodiments, the respirable dry particles of the inventioncan contain (a) a calcium salt selected from calcium lactate, calciumcitrate or calcium sulfate in an amount of at least about 30%, at leastabout 40%, at least about 45% by weight, or at least about 50% by weightof dry particle; and (b) a sodium salt, such as sodium chloride, in anamount of at least about 25% or at least about 30% by weight of dryparticle, and have any of the properties or features described herein.If desired, an excipient, such as leucine, maltodextrin, mannitol or anycombination thereof, can be present an amount of about 50% or less orabout 20% or less by weight of the dry particle. For example, therespirable dry particles of the invention can include (a) a calcium saltin an amount of about 30% to about 65%, about 40% to about 65%, or about45% to about 65% by weight of dry particle; (b) a sodium salt, such assodium chloride, in an amount of about 25% to about 60%, or about 30% toabout 60% by weight of dry particle; (c) an excipient, such as leucine,maltodextrin, mannitol or any combination thereof, in an amount of about20% or less by weight of dry particle, or more preferably about 10% orless by weight of dry particle, and (d) have any of the properties orfeatures, such as 1/4 bar, 0.5/4 bar, VMGD, MMAD, FPF described herein.

In some aspects, the respirable dry particles comprise a divalent metalion salt and a monovalent salt and are characterized by the crystallineand amorphous content of the particles. For example, the respirable dryparticles can comprise a mixture of amorphous and crystalline content,such as an amorphous divalent metal ion salt-rich phase and acrystalline monovalent salt phase. Respirable dry particles of this typeprovide several advantages. For example as described herein, thecrystalline phase (e.g. crystalline sodium chloride) can contribute tothe stability of the dry particle in the dry state and to thedispersibility characteristics, whereas the amorphous phase (e.g.,amorphous calcium salt) can facilitate rapid water uptake anddissolution of the particle upon deposition in the respiratory tract. Itis particularly advantageous when salts with relatively high aqueoussolubilities (such as sodium chloride) that are present in the dryparticles are in a crystalline state and when salts with relatively lowaqueous solubilities (such as calcium citrate) are present in the dryparticles in an amorphous state.

The amorphous phase is also characterized by a high glass transitiontemperature (T_(g)), such as a T_(g) of at least 100° C., at least 110°C., 120° C., at least 125° C., at least 130° C., at least 135° C., atleast 140° C., between 120° C. and 200° C., between 125° C. and 200° C.,between 130° C. and 200° C., between 120° C. and 190° C., between 125°C. and 190° C., between 130° C. and 190° C., between 120° C. and 180°C., between 125° C. and 180° C., or between 130° C. and 180° C.

In some embodiments, the respirable dry particles contain divalent metalcation salt-rich amorphous phase and a monovalent salt crystalline phaseand the ratio of amorphous phase to crystalline phase (w:w) is about5:95 to about 95:5, about 5:95 to about 10:90, about 10:90 to about20:80, about 20:80 to about 30:70, about 30:70 to about 40:60, about40:60 to about 50:50; about 50:50 to about 60:40, about 60:40 to about70:30, about 70:30 to about 80:20, or about 90:10 to about 95:5. Inother embodiments, the respirable dry particles contain divalent metalcation salt-rich amorphous phase and a monovalent salt crystalline phaseand the ratio of amorphous phase to particle by weight (w:w) is about5:95 to about 95:5, about 5:95 to about 10:90, about 10:90 to about20:80, about 20:80 to about 30:70, about 30:70 to about 40:60, about40:60 to about 50:50; about 50:50 to about 60:40, about 60:40 to about70:30, about 70:30 to about 80:20, or about 90:10 to about 95:5. Inother embodiments, the respirable dry particles contain divalent metalcation salt-rich amorphous phase and a monovalent salt crystalline phaseand the ratio of crystalline phase to particle by weight (w:w) is about5:95 to about 95:5, about 5:95 to about 10:90, about 10:90 to about20:80, about 20:80 to about 30:70, about 30:70 to about 40:60, about40:60 to about 50:50; about 50:50 to about 60:40, about 60:40 to about70:30, about 70:30 to about 80:20, or about 90:10 to about 95:5.

In some embodiments, the respirable dry particles comprises a calciumsalt, such as calcium citrate, calcium sulfate, calcium lactate, calciumchloride or any combination thereof, and a sodium salt, such as sodiumchloride, sodium citrate, sodium sulfate, sodium lactate, or anycombination thereof, wherein the respirable dry particle contains ancalcium salt-rich amorphous phase, and a crystalline sodium salt phase.In particular embodiments, the calcium salt-rich amorphous phaseincludes calcium citrate and at least some calcium chloride, calciumlactate and at least some calcium chloride, or calcium sulfate and atleast some calcium chloride. In some embodiments, the respirable dryparticles contain calcium salt-rich amorphous phase and a sodium saltcrystalline phase and the ratio of amorphous phase to crystalline phase(w:w) is about 5:95 to about 95:5, about 5:95 to about 10:90, about10:90 to about 20:80, about 20:80 to about 30:70, about 30:70 to about40:60, about 40:60 to about 50:50; about 50:50 to about 60:40, about60:40 to about 70:30, about 70:30 to about 80:20, or about 90:10 toabout 95:5. In other embodiments, the respirable dry particles containcalcium salt-rich amorphous phase and a sodium salt crystalline phaseand the ratio of amorphous phase to particle by weight (w:w) is about5:95 to about 95:5, about 5:95 to about 10:90, about 10:90 to about20:80, about 20:80 to about 30:70, about 30:70 to about 40:60, about40:60 to about 50:50; about 50:50 to about 60:40, about 60:40 to about70:30, about 70:30 to about 80:20, or about 90:10 to about 95:5. Inother embodiments, the respirable dry particles contain calciumsalt-rich amorphous phase and a sodium salt crystalline phase and theratio of crystalline phase to particle by weight (w:w) is about 5:95 toabout 95:5, about 5:95 to about 10:90, about 10:90 to about 20:80, about20:80 to about 30:70, about 30:70 to about 40:60, about 40:60 to about50:50; about 50:50 to about 60:40, about 60:40 to about 70:30, about70:30 to about 80:20, or about 90:10 to about 95:5.

Preferrably, the respirable dry particles have a 1/4 bar or 0.5/4 bar of2 or less, as described herein. For example, a 1/4 bar or 0.5/4 of 1.9or less, 1.8 or less, 1.7 or less, 1.6 or less, 1.5 or less, 1.4 orless, 1.3 or less, 1.2 or less, 1.1 or less or about 1.0. Alternativelyor in addition, the respirable dry particles have an MMAD of about 5microns or less. Alternatively or in addition, the respirable dryparticles can have a VMGD between about 0.5 microns and about 5 microns,or a VMGD between about 5 microns and about 20 microns. Alternatively orin addition, the respirable dry particles can have a heat of solutionthat not is greater than about −10 kcal/mol (e.g., between −10 kcal/moland 10 kcal/mol).

As described herein, the respirable dry particles can further comprisean excipient, such as leucine, maltodextrin or mannitol. The excipientcan be crystalline or amorphous or present in a combination of theseforms. In some embodiments, the excipient is amorphous or predominatelyamorphous.

As described herein, RAMAN spectra of respirable dry powders thatcontained an excipient (i.e., leucine, maltodextrin) did not includepeaks assigned to the excipients. This indicates that the excipientswere not concentrated at the surface of the particles, and that theexcipients are either evenly distributed throughout the particle or notexposed to the surface of the particle. Leucine excipients, inparticular, have been reported to improve dispersibility whenconcentrated on the surface of particles. See, e.g., US2003/0186894.Accordingly, it does not appear that leucine is acting as a dispersionenhancer in this way. Thus, in the respirable dry particles of theinvention that contain an excipient (e.g., leucine), the excipient canbe distributed within the particle but not on the particle surface, ordistributed throughout the particle (e.g., homogenously distributed).For example, in some particular embodiments, a resperable dry particleof the invention does not produce a characteristic peak indicative ofthe presence of an excipient (e.g., leucine) under RAMAN spectroscopy.In more particular embodiments, a dry respirable powder that containsleucine does not produce a characteristic leucine peak (e.g., at 1340cm⁻¹) under RAMAN spectroscopy.

As described herein, some powders of the invention have poor flowproperties. Yet, surprisingly, these powders are highly dispersible.This is surprising because flow properties and dispersibility are bothknown to be negatively effected by particle agglomeration oraggregation. Thus, it was unexpected that particles that have poor flowcharacteristics would be highly dispersible.

In addition to any of the features and properties described herein, inany combination, the respirable dry particles can have poor flowproperties yet have good dispersibility. For example, the respirable dryparticles can have a Hausner Ratio that is greater than 1.35 (e.g, 1.4or greater, 1.5 or greater, 1.6 or greater, 1.7 or greater, 1.8 orgreater, 1.9 or greater, 2.0 or greater) and also have a 1/4 bar or 0.5bar that is 2 or less, 1.9 or less, 1.8 or less, 1.7 or less, 1.6 orless, 1.5 or less, 1.4 or less, 1.3 or less, 1.2 or less, 1.1 or less orabout 1.0.

In addition to any of the features and properties described herein, inany combination, the respirable dry particles can have a heat ofsolution that is not highly exothermic. Preferably, the heat of solutionis determined using the ionic liquid of a simulated lung fluid (e.g. asdescribed in Moss, O. R. 1979. Simulants of lung interstitial fluid.Health Phys. 36, 447-448; or in Sun, G. 2001. Oxidative interactions ofsynthetic lung epithelial lining fluid with metal-containing particulatematter. Am J Physiol Lung Cell Mol Physiol. 281, L807-L815) at pH 7.4and 37° C. in an isothermal calorimeter. For example, the respirable dryparticles can have a heat of solution that is less exothermic than theheat of solution of calcium chloride dihydratedihydrate, e.g., have aheat of solution that is greater than about −10 kcal/mol, greater thanabout −9 kcal/mol, greater than about −8 kcal/mol, greater than about −7kcal/mol, greater than about −6 kcal/mol, greater than about −5kcal/mol, greater than about −4 kcal/mol, greater than about −3kcal/mol, greater than about −2 kcal/mol, greater than about −1 kcal/molor about −10 kcal/mol to about 10 kcal/mol.

If desired, the salt formulation can include one or more additionalagents, such as mucoactive or mucolytic agents, surfactants,antibiotics, antivirals, antihistamines, cough suppressants,bronchodilators, anti-inflammatory agents, steroids, vaccines,adjuvants, expectorants, macromolecules, therapeutics that are helpfulfor chronic maintenance of CF.

Examples of suitable mucoactive or mucolytic agents include MUC5AC andMUC5B mucins, DNA-ase, N-acetylcysteine (NAC), cysteine, nacystelyn,dornase alfa, gelsolin, heparin, heparin sulfate, P2Y2 agonists (e.g.UTP, INS365), hypertonic saline, and mannitol.

Suitable surfactants include L-alpha-phosphatidylcholine dipalmitoyl(“DPPC”), diphosphatidyl glycerol (DPPG),1,2-Dipalmitoyl-sn-glycero-3-phospho-L-serine (DPPS),1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DSPC),1,2-Distearoyl-sn-glycero-3-phosphoethanolamine (DSPE),1-palmitoyl-2-oleoylphosphatidylcholine (POPC), fatty alcohols,polyoxyethylene-9-lauryl ether, surface active fatty, acids, sorbitantrioleate (Span 85), glycocholate, surfactin, poloxomers, sorbitan fattyacid esters, tyloxapol, phospholipids, and alkylated sugars.

If desired, the salt formulation can contain an antibiotic. For example,salt formulations for treating bacterial pneumonia or VAT, can furthercomprise an antibiotic, such as a macrolide (e.g., azithromycin,clarithromycin and erythromycin), a tetracycline (e.g., doxycycline,tigecycline), a fluoroquinolone (e.g., gemifloxacin, levofloxacin,ciprofloxacin and mocifloxacin), a cephalosporin (e.g., ceftriaxone,defotaxime, ceftazidime, cefepime), a penicillin (e.g., amoxicillin,amoxicillin with clavulanate, ampicillin, piperacillin, and ticarcillin)optionally with a β-lactamase inhibitor (e.g., sulbactam, tazobactam andclavulanic acid), such as ampicillin-sulbactam, piperacillin-tazobactamand ticarcillin with clavulanate, an aminoglycoside (e.g., amikacin,arbekacin, gentamicin, kanamycin, neomycin, netilmicin, paromomycin,rhodostreptomycin, streptomycin, tobramycin, and apramycin), a penem orcarbapenem (e.g. doripenem, ertapenem, imipenem and meropenem), amonobactam (e.g., aztreonam), an oxazolidinone (e.g., linezolid),vancomycin, glycopeptide antibiotics (e.g. telavancin),tuberculosis-mycobacterium antibiotics and the like.

If desired, the salt formulation can contain an agent for treatinginfections with mycobacteria, such as Mycobacterium tuberculosis.Suitable agents for treating infections with mycobacteria (e.g., M.tuberculosis) include an aminoglycoside (e.g. capreomycin, kanamycin,streptomycin), a fluoroquinolone (e.g. ciprofloxacin, levofloxacin,moxifloxacin), isozianid and isozianid analogs (e.g. ethionamide),aminosalicylate, cycloserine, diarylquinoline, ethambutol, pyrazinamide,protionamide, rifampin, and the like.

If desired, the salt formulation can contain a suitable antiviral agent,such as oseltamivir, zanamavir, amantidine, rimantadine, ribavirin,gancyclovir, valgancyclovir, foscavir, Cytogam® (Cytomegalovirus ImmuneGlobulin), pleconaril, rupintrivir, palivizumab, motavizumab,cytarabine, docosanol, denotivir, cidofovir, and acyclovir. The saltformulation can contain a suitable anti-influenza agent, such aszanamivir, oseltamivir, amantadine, or rimantadine.

Suitable antihistamines include clemastine, asalastine, loratadine,fexofenadine and the like.

Suitable cough suppressants include benzonatate, benproperine,clobutinal, diphenhydramine, dextromethorphan, dibunate, fedrilate,glaucine, oxalamine, piperidione, opioids such as codeine and the like.

Suitable bronchodilators include short-acting beta₂ agonists,long-acting beta₂ agonists (LABA), long-acting muscarinic anagonists(LAMA), combinations of LABAs and LAMAs, methylxanthines, and the like.Suitable short-active beta2 agonists include albuterol, epinephrine,pirbuterol, levalbuterol, metaproteronol, maxair, and the like. SuitableLABAs include salmeterol, formoterol and isomers (e.g. arformoterol),clenbuterol, tulobuterol, vilanterol (Revolair™), indacaterol, and thelike. Examples of LAMAs include tiotroprium, glycopyrrolate, aclidinium,ipratropium and the like. Examples of combinations of LABAs and LAMAsinclude indacaterol with glycopyrrolate, indacaterol with tiotropium,and the like. Examples of methylxanthine include theophylline, and thelike.

Suitable anti-inflammatory agents include leukotriene inhibitors, PDE4inhibitors, other anti-inflammatory agents, and the like. Suitableleukotriene inhibitors include montelukast (cystinyl leukotrieneinhibitors), masilukast, zafirleukast (leukotriene D4 and E4 receptorinhibitors), zileuton (5-lipoxygenase inhibitors), and the like.Suitable PDE4 inhibitors include cilomilast, roflumilast, and the like.Other anti-inflammatory agents include omalizumab (anti IgEimmunoglobulin), IL-13 and IL-13 receptor inhibitors (such as AMG-317,MILR1444A, CAT-354, QAX576, IMA-638, Anrukinzumab, IMA-026, MK-6105,DOM-0910 and the like), IL-4 and IL-4 receptor inhibitors (such asPitrakinra, AER-003,AIR-645, APG-201, DOM-0919 and the like) IL-1inhibitors such as canakinumab, CRTh2 receptor antagonists such asAZD1981 (from AstraZeneca), neutrophil elastase inhibitor such asAZD9668 (from AstraZeneca), P38 kinase inhibitor such as losmapimed, andthe like.

Suitable steroids include corticosteroids, combinations ofcorticosteroids and LABAs, combinations of corticosteroids and LAMAs,and the like. Suitable corticosteroids include budesonide, fluticasone,flunisolide, triamcinolone, beclomethasone, mometasone, ciclesonide,dexamethasone, and the like. Combinations of corticosteroids and LABAsinclude salmeterol with fluticasone, formoterol with budesonide,formoterol with fluticasone, formoterol with mometasone, indacaterolwith mometasone, and the like.

Suitable expectorants include guaifenesin, guaiacolculfonate, ammoniumchloride, potassium iodide, tyloxapol, antimony pentasulfide and thelike.

Suitable vaccines such as nasally inhaled influenza vaccines and thelike.

Suitable macromolecules include proteins and large peptides,polysaccharides and oligosaccharides, and DNA and RNA nucleic acidmolecules and their analogs having therapeutic, prophylactic ordiagnostic activities. Proteins can include antibodies such asmonoclonal antibodies. Nucleic acid molecules include genes, antisensemolecules such as siRNAs that bind to complementary DNA, RNA, orribosomes to inhibit transcription or translation.

Selected macromolecule drugs for systemic applications: Calcitonin,Erythropoietin (EPO), Factor IX, Granulocyte Colony Stimulating Factor(G-CSF), Granulocyte Macrophage Colony, Stimulating Factor (GM-CSF),Growth Hormone, Insulin, Interferon Alpha, Interferon Beta, InterferonGamma, Luteinizing Hormone Releasing Hormone (LHRH), folliclestimulating hormone (FSH), Ciliary Neurotrophic Factor, Growth HormoneReleasing Factor (GRF), Insulin-Like Growth Factor, Insulinotropin,Interleukin-1 Receptor Antagonist, Interleukin-3, Interleukin-4,Interleukin-6, Macrophage Colony Stimulating Factor (M-CSF), ThymosinAlpha 1, IIb/IIIa Inhibitor, Alpha-1 Antitrypsin, Anti-RSV Antibody,palivizumab, motavizumab, and ALN-RSV, Cystic Fibrosis TransmembraneRegulator (CFTR) Gene, Deoxyribonuclase (DNase), Heparin,Bactericidal/Permeability Increasing Protein (BPI), Anti-Cytomegalovirus(CMV) Antibody, Interleukin-1 Receptor Antagonist, and the like.

Selected therapeutics that are helpful for chronic maintenance of CFinclude antibiotics/macrolide antibiotics, bronchodilators, inhaledLABAs, and agents to promote airway secretion clearance. Suitableexamples of antibiotics/macrolide antibiotics include tobramycin,azithromycin, ciprofloxacin, colistin, and the like. Suitable examplesof bronchodilators include inhaled short-acting beta₂ agonists such asalbuterol, and the like. Suitable examples of inhaled LABAs includesalmeterol, formoterol, and the like. Suitable examples of agents topromote airway secretion clearance include dornase alfa, hypertonicsaline, and the like.

It is generally preferred that the respirable dry particles and drypowders do not contain salts, excipients, or other active ingredientsthat have a molecular weight of greater than about 1 kilodalton (1000dalton, Da). For example, the respirable particles of the inventionpreferably do not contain a protein, a polypeptide, oligopeptides,nucleic acid or an oligonucleotide with a molecular weight of greaterthan 1 KDa, great than about 900 Da, greater than about 800 Da, greaterthan about 700 Da, or greater than about 600 Da.

Because the respirable dry powders and respirable dry particlesdescribed herein contain salts, they may be hygroscopic. Accordingly itis desirable to store or maintain the respirable dry powders andrespirable dry particles under conditions to prevent hydration of thepowders. For example, if it is desirable to prevent hydration, therelative humidity of the storage environment should be less than 75%,less than 60%, less than 50%, less than 40%, less than 30%, less than25%, less than 20%, less than 15%, less than 10%, or less than 5%humidity. The respirable dry powders and respirable dry particles can bepackaged (e.g., in sealed capsules, blisters, vials) under theseconditions.

The invention also relates to respirable dry powders or respirable dryparticles produced by preparing a feedstock solution, emulsion orsuspension and spray drying the feedstock according to the methodsdescribed herein. The feedstock can be prepared using (a) a calciumsalt, such as calcium lactate or calcium chloride, in an amount of atleast about 25% by weight (e.g., of total solutes used for preparing thefeedstock) and (b) a sodium salt, such as sodium citrate, sodiumchloride or sodium sulfate, in an amount of at least about 1% by weight(e.g., of total solutes used for preparing the feedstock). If desired,one or more excipient, such as leucine can be added to the feedstock inan amount of about 74% or less by weight (e.g., of total solutes usedfor preparing the feedstock). For example, the calcium salt used forpreparing the feedstock can be in an amount of at least about 30%, atleast about 35%, at least about 40%, at least about 50%, at least about60% or at least about 70% by weight of total solutes used for preparingthe feedstock. The sodium salt used for preparing the feedstock, forexample, can be in an amount of at least about 2%, at least about 3%, atleast about 4%, at least about 5%, at least about 6%, at least about 7%,at least about 8%, at least about 9%, at least about 10%, at least about20%, at least about 25%, at least about 30%, at least about 40%, atleast about 50%, at least about 55% or at least about 65% by weight oftotal solutes used for preparing the feedstock. The excipient added tothe feedstock, for example, can be in an amount about 50% or less, about30% or less, about 20% or less, about 10% or less, about 9% or less,about 8% or less, about 7% or less, about 6% or less, about 5% or less,about 4% or less, about 3% or less, about 2% or less, about 1% or lessby weight of total solutes used for preparing the feedstock.

In an embodiment, the respirable dry powders or respirable dry particlesof the invention can be obtained by (1) preparing a feedstock comprising(a) a dry solute containing in percent by weight of the total dry soluteabout 10.0% leucine, about 35.1% calcium chloride and about 54.9% sodiumcitrate and (a) one or more suitable solvents for dissolution of thesolute and formation of the feedstock, and (2) spray drying thefeedstock. In another embodiment, the respirable dry powders orrespirable dry particles of the invention can be obtained by (1)preparing a feedstock comprising (a) a dry solute containing in percentby weight of the total dry solute about 10.0% leucine, about 58.6%calcium lactate and about 31.4% sodium chloride and (a) one or moresuitable solvents for dissolution of the solute and formation of thefeedstock, and (2) spray drying the feedstock. In another embodiment,the respirable dry powders or respirable dry particles of the inventioncan be obtained by (1) preparing a feedstock comprising (a) a dry solutecontaining in percent by weight of the total dry solute about 10.0%leucine, about 39.6% calcium chloride and about 50.44% sodium sulfateand (b) one or more suitable solvents for dissolution of the solute andformation of the feedstock and (2) spray drying the feedstock. Inanother embodiment, the respirable dry powders or respirable dryparticles of the invention can be obtained by (1) preparing a feedstockcomprising (a) a dry solute containing in percent by weight of the totaldry solute about 10.0% maltodextrin, about 58.6% calcium lactate andabout 31.4% sodium chloride and (a) one or more suitable solvents fordissolution of the solute and formation of the feedstock, and (2) spraydrying the feedstock. As described herein, various methods (e.g., staticmixing, bulk mixing) can be used for mixing the solutes and solvents toprepare feedstocks, which are known in the art. If desired, othersuitable methods of mixing may be used. For example, additionalcomponents that cause or facilitate the mixing can be included in thefeedstock. For example, carbon dioxide produces fizzing or effervescenceand thus can serve to promote physical mixing of the solute andsolvents. Various salts of carbonate or bicarbonate can promote the sameeffect that carbon dioxide produces and, therefore, can be used inpreparation of the feedstocks of the invention.

In preferred embodiments, the respirable dry powders or respirable dryparticles of the invention possess aerosol characteristics that permiteffective delivery of the respirable dry particles to the respiratorysystem without the use of propellants.

In an embodiment, the respirable dry powders or respirable dry particlesof the invention can be produced through an ion exchange reaction. Incertain embodiments of the invention, two saturated or sub-saturatedsolutions are fed into a static mixer in order to obtain a saturated orsupersaturated solution post-static mixing. Preferably, the post-mixedsolution is supersaturated. The two solutions may be aqueous or organic,but are preferably substantially aqueous. The post-static mixingsolution is then fed into the atomizing unit of a spray dryer. In apreferable embodiment, the post-static mixing solution is immediatelyfed into the atomizer unit. Some examples of an atomizer unit include atwo-fluid nozzle, a rotary atomizer, or a pressure nozzle. Preferably,the atomizer unit is a two-fluid nozzle. In one embodiment, thetwo-fluid nozzle is an internally mixing nozzle, meaning that the gasimpinges on the liquid feed before exiting to most outward orifice. Inanother embodiment, the two-fluid nozzle is an externally mixing nozzle,meaning that the gas impinges on the liquid feed after exiting the mostoutward orifice.

The dry particles of the invention can be blended with an activeingredient or co-formulated with an active ingredient to maintaincharacteristic high dispersibility of the dry particles and dry powdersof the invention.

In one aspect, salts of divalent cations (e.g., calcium, magnesium) canbe co-formulated with a non-calcium active agent, to make small, highlydispersible powders or large, porous particles. Optionally, theseparticles may include a monovalent cationic salt (e.g., sodium,potassium), and also optionally an excipient (e.g., leucine,maltodextrin, mannitol, lactose). The components can be mixed (e.g.,mixed as one solution, static mixed as two solutions) together in asingle particle before spray drying.

In another aspect, the dry particles of the invention are large, porous,and are dispersible. The size of the dry particles can be expressed in avariety of ways. The particles may have VMGD between 5 to 30 μm, orbetween 5 and 20 μm, with a tap density of less than 0.5 g/cc,preferably less than 0.4 g/cc.

Methods for Preparing Dry Powders and Dry Particles

The respirable dry particles and dry powders can be prepared using anysuitable method. Many suitable methods for preparing respirable drypowders and particles are conventional in the art, and include singleand double emulsion solvent evaporation, spray drying, milling (e.g.,jet milling), blending, solvent extraction, solvent evaporation, phaseseparation, simple and complex coacervation, interfacial polymerization,suitable methods that involve the use of supercritical carbon dioxide(CO₂), and other suitable methods. Respirable dry particles can be madeusing methods for making microspheres or microcapsules known in the art.These methods can be employed under conditions that result in theformation of respirable dry particles with desired aerodynamicproperties (e.g., aerodynamic diameter and geometric diameter). Ifdesired, respirable dry particles with desired properties, such as sizeand density, can be selected using suitable methods, such as sieving.

The respirable dry particles are preferably spray dried. Suitablespray-drying techniques are described, for example, by K. Masters in“Spray Drying Handbook”, John Wiley & Sons, New York (1984). Generally,during spray-drying, heat from a hot gas such as heated air or nitrogenis used to evaporate a solvent from droplets formed by atomizing acontinuous liquid feed. If desired, the spray drying or otherinstruments, e.g., jet milling instrument, used to prepare the dryparticles can include an inline geometric particle sizer that determinesa geometric diameter of the respirable dry particles as they are beingproduced, and/or an inline aerodynamic particle sizer that determinesthe aerodynamic diameter of the respirable dry particles as they arebeing produced.

For spray drying, solutions, emulsions or suspensions that contain thecomponents of the dry particles to be produced in a suitable solvent(e.g., aqueous solvent, organic solvent, aqueous-organic mixture oremulsion) are distributed to a drying vessel via an atomization device.For example, a nozzle or a rotary atomizer may be used to distribute thesolution or suspension to the drying vessel. For example, a rotaryatomizer having a 4- or 24-vaned wheel may be used. Examples of suitablespray dryers that can be outfitted with either a rotary atomizer or anozzle, include, Mobile Minor Spray Dryer or the Model PSD-1, bothmanufactured by Niro, Inc. (Denmark). Actual spray drying conditionswill vary depending, in part, on the composition of the spray dryingsolution or suspension and material flow rates. The person of ordinaryskill will be able to determine appropriate conditions based on thecompositions of the solution, emulsion or suspension to be spray dried,the desired particle properties and other factors. In general, the inlettemperature to the spray dryer is about 100° C. to about 300° C., andpreferably is about 220° C. to about 285° C. The spray dryer outlettemperature will vary depending upon such factors as the feedtemperature and the properties of the materials being dried. Generally,the outlet temperature is about 50° C. to about 150° C., preferablyabout 90° C. to about 120° C., or about 98° C. to about 108° C. Ifdesired, the respirable dry particles that are produced can befractionated by volumetric size, for example, using a sieve, orfractioned by aerodynamic size, for example, using a cyclone, and/orfurther separated according to density using techniques known to thoseof skill in the art.

To prepare the respirable dry particles of the invention, generally, asolution, emulsions or suspension that contains the desired componentsof the dry powder (i.e., a feed stock) is prepared and spray dried undersuitable conditions. Preferably, the dissolved or suspended solidsconcentration in the feed stock is at least about 1 g/L, at least about2 g/L, at least about 5 g/L, at least about 10 g/L, at least about 15g/L, at least about 20 g/L, at least about 30 g/L, at least about 40g/L, at least about 50 g/L, at least about 60 g/L, at least about 70g/L, at least about 80 g/L, at least about 90 g/L, or at least about 100g/L. The feed stock can be provided by preparing a single solution orsuspension by dissolving or suspending suitable components (e.g., salts,excipients, other active ingredients) in a suitable solvent. Thesolvent, emulsion or suspension can be prepared using any suitablemethods, such as bulk mixing of dry and/or liquid components or staticmixing of liquid components to form a combination. For example, ahydrophillic component (e.g., an aqueous solution) and a hydrophobiccomponent (e.g., an organic solution) can be combined using a staticmixer to form a combination. The combination can then be atomized toproduce droplets, which are dried to form respirable dry particles.Preferably, the atomizing step is performed immediately after thecomponents are combined in the static mixer.

In one example, respirable dry particles that contain calcium citrate,sodium chloride and leucine are prepared by spray drying. A first phaseis prepared that comprises an aqueous solution of sodium citrate andleucine. A second phase is prepared that comprises calcium chloride inan appropriate solvent. One or both solutions may be separately heatedas needed to assure solubility of their components. The first and secondphases are then combined in a static mixer to form a combination. Thecombination is spray dried to form respirable dry particles.

The feed stock, or components of the feed stock, can be prepared usingany suitable solvent, such as an organic solvent, an aqueous solvent ormixtures thereof. Suitable organic solvents that can be employed includebut are not limited to alcohols such as, for example, ethanol, methanol,propanol, isopropanol, butanols, and others. Other organic solventsinclude but are not limited to perfluorocarbons, dichloromethane,chloroform, ether, ethyl acetate, methyl tert-butyl ether and others.Co-solvents that can be employed include an aqueous solvent and anorganic solvent, such as, but not limited to, the organic solvents asdescribed above. Aqueous solvents include water and buffered solutions.

The feed stock or components of the feed stock can have any desired pH,viscosity or other properties. If desired, a pH buffer can be added tothe solvent or co-solvent or to the formed mixture. Generally, the pH ofthe mixture ranges from about 3 to about 8.

Respirable dry particles and dry powders can be fabricated and thenseparated, for example, by filtration or centrifugation by means of acyclone, to provide a particle sample with a preselected sizedistribution. For example, greater than about 30%, greater than about40%, greater than about 50%, greater than about 60%, greater than about70%, greater than about 80%, or greater than about 90% of the respirabledry particles in a sample can have a diameter within a selected range.The selected range within which a certain percentage of the respirabledry particles fall can be, for example, any of the size ranges describedherein, such as between about 0.1 to about 3 microns VMGD.

The diameter of the respirable dry particles, for example, their VMGD,can be measured using an electrical zone sensing instrument such as aMultisizer lie, (Coulter Electronic, Luton, Beds, England), or a laserdiffraction instrument such as a HELOS system (Sympatec, Princeton,N.J.). Other instruments for measuring particle geometric diameter arewell known in the art. The diameter of respirable dry particles in asample will range depending upon factors such as particle compositionand methods of synthesis. The distribution of size of respirable dryparticles in a sample can be selected to permit optimal depositionwithin targeted sites within the respiratory system.

Experimentally, aerodynamic diameter can be determined using time offlight (TOF) measurements. For example, an instrument such as the Model3225 Aerosizer DSP Particle Size Analyzer (Amherst Process Instrument,Inc., Amherst, Mass.) can be used to measure aerodynamic diameter. TheAerosizer measures the time taken for individual respirable dryparticles to pass between two fixed laser beams.

Aerodynamic diameter also can be experimentally determined directlyusing conventional gravitational settling methods, in which the timerequired for a sample of respirable dry particles to settle a certaindistance is measured. Indirect methods for measuring the mass medianaerodynamic diameter include the Andersen Cascade Impactor and themulti-stage liquid impinger (MSLI) methods. The methods and instrumentsfor measuring particle aerodynamic diameter are well known in the art.

Tap density is a measure of the envelope mass density characterizing aparticle. The envelope mass density of a particle of a statisticallyisotropic shape is defined as the mass of the particle divided by theminimum sphere envelope volume within which it can be enclosed. Featureswhich can contribute to low tap density include irregular surfacetexture and porous structure. Tap density can be measured by usinginstruments known to those skilled in the art such as the Dual PlatformMicroprocessor Controlled Tap Density Tester (Vankel, N.C.), aGeoPyc™instrument (Micrometrics Instrument Corp., Norcross, Ga.), orSOTAX Tap Density Tester model TD2 (SOTAX Corp., Horsham, Pa.). Tapdensity can be determined using the method of USP Bulk Density andTapped Density, United States Pharmacopia convention, Rockville, Md.,10^(th) Supplement, 4950-4951, 1999.

Fine particle fraction can be used as one way to characterize theaerosol performance of a dispersed powder. Fine particle fractiondescribes the size distribution of airborne respirable dry particles.Gravimetric analysis, using a Cascade impactor, is one method ofmeasuring the size distribution, or fine particle fraction, of airbornerespirable dry particles. The Andersen Cascade Impactor (ACI) is aneight-stage impactor that can separate aerosols into nine distinctfractions based on aerodynamic size. The size cutoffs of each stage aredependent upon the flow rate at which the ACI is operated. The ACI ismade up of multiple stages consisting of a series of nozzles (i.e., ajet plate) and an impaction surface (i.e., an impaction disc). At eachstage an aerosol stream passes through the nozzles and impinges upon thesurface. Respirable dry particles in the aerosol stream with a largeenough inertia will impact upon the plate. Smaller respirable dryparticles that do not have enough inertia to impact on the plate willremain in the aerosol stream and be carried to the next stage. Eachsuccessive stage of the ACI has a higher aerosol velocity in the nozzlesso that smaller respirable dry particles can be collected at eachsuccessive stage.

If desired, a two-stage collapsed ACI can also be used to measure fineparticle fraction. The two-stage collapsed ACI consists of only the toptwo stages of the eight-stage ACI and allows for the collection of twoseparate powder fractions. Specifically, a two-stage collapsed ACI iscalibrated so that the fraction of powder that is collected on stage oneis composed of respirable dry particles that have an aerodynamicdiameter of less than 5.6 microns and greater than 3.4 microns. Thefraction of powder passing stage one and depositing on a collectionfilter is thus composed of respirable dry particles having anaerodynamic diameter of less than 3.4 microns. The airflow at such acalibration is approximately 60 L/min.

The FPF (<5.6) has been demonstrated to correlate to the fraction of thepowder that is able to make it into the lung of the patient, while theFPF (<3.4) has been demonstrated to correlate to the fraction of thepowder that reaches the deep lung of a patient. These correlationsprovide a quantitative indicator that can be used for particleoptimization.

An ACI can be used to approximate the emitted dose, which herein iscalled gravimetric recovered dose and analytical recovered dose.“Gravimetric recovered dose” is defined as the ratio of the powderweighed on all stage filters of the ACI to the nominal dose. “Analyticalrecovered dose” is defined as the ratio of the powder recovered fromrinsing all stages, all stage filters, and the induction port of the ACIto the nominal dose. The FPF_TD (<5.0) is the ratio of the interpolatedamount of powder depositing below 5.0 μm on the ACI to the nominal dose.The FPF_RD (<5.0) is the ratio of the interpolated amount of powderdepositing below 5.0 μm on the ACI to either the gravimetric recovereddose or the analytical recovered dose.

Another way to approximate emitted dose is to determine how much powderleaves its container, e.g. capture or blister, upon actuation of a drypowder inhaler (DPI). This takes into account the percentage leaving thecapsule, but does not take into account any powder depositing on theDPI. The emitted dose is the ratio of the weight of the capsule with thedose before inhaler actuation to the weight of the capsule after inhaleractuation. This measurement can also be called the capsule emittedpowder mass (CEPM)

A Multi-Stage Liquid Impinger (MSLI) is another device that can be usedto measure fine particle fraction. The Multi-stage liquid Impingeroperates on the same principles as the ACI, although instead of eightstages, MSLI has five. Additionally, each MSLI stage consists of anethanol-wetted glass frit instead of a solid plate. The wetted stage isused to prevent particle bounce and re-entrainment, which can occur whenusing the ACI.

The invention also relates to a method for producing a respirable drypowder comprising respirable dry particles that contain calcium citrateor calcium sulfate. The method comprises a) providing a first liquidfeed stock comprising an aqueous solution of calcium chloride, and asecond liquid feed stock comprising an aqueous solution of sodiumsulfate or sodium citrate; b) mixing the first liquid feed stock and thesecond liquid feed stock to produce a mixture in which an anion exchangereaction occurs to produce a saturated or supersaturated solutioncomprising calcium sulfate and sodium chloride, or calcium citrate andsodium chloride; and c) spray drying the saturated or supersaturatedsolution produced in b) to produce respirable dry particles. The firstliquid feed stock and the second liquid feed stock can be batch mixed orpreferably, static mixed. In some embodiments, the resulting mixture isspray dried, and atomized within 60 minutes, within 30 minutes, within15 minutes, within 10 minutes, within 5 minutes, within 4 minutes,within 3 minutes, within 2 minutes, within 1 minute, within 45 seconds,within 30 seconds, within 15 seconds, within 5 seconds of mixing,preferably static mixing.

The invention also relates to a respirable dry powder or respirable dryparticles produced using any of the methods described herein.

The respirable dry particles of the invention can also be characterizedby the chemical stability of the salts or the excipients that therespirable dry particles comprise. The chemical stability of theconstituent salts can affect important characteristics of the respirableparticles including shelf-life, proper storage conditions, acceptableenvironments for administration, biological compatibility, andeffectiveness of the salts. Chemical stability can be assessed usingtechniques well known in the art. One example of a technique that can beused to assess chemical stability is reverse phase high performanceliquid chromatography (RP-HPLC). Respirable dry particles of theinvention include salts that are generally stable over a long periodtime.

If desired, the respirable dry particles and dry powders describedherein can be further processed to increase stability. An importantcharacteristic of pharmaceutical dry powders is whether they are stableat different temperature and humidity conditions. Unstable powders willabsorb moisture from the environment and agglomerate, thus alteringparticle size distribution of the powder.

Excipients, such as maltodextrin, may be used to create more stableparticles and powders. The maltodextrin may act as an amorphous phasestabilizer and inhibit the components from converting from an amorphousto crystalline state. Alternatively, a post-processing step to help theparticles through the crystallization process in a controlled way (e.g.,on the baghouse at elevated humidity) can be employed with the resultantpowder potentially being further processed to restore theirdispersibility if agglomerates formed during the crystallizationprocess, such as by passing the particles through a cyclone to breakapart the agglomerates. Another possible approach is to optimize aroundprocess conditions that lead to manufacturing particles that are morecrystalline and therefore more stable. Another approach is to usedifferent excipients, or different levels of current excipients toattempt to manufacture more stable forms of the salts.

The respirable dry particles and dry powders described herein aresuitable for inhalation therapies. The respirable dry particles may befabricated with the appropriate material, surface roughness, diameterand tap density for localized delivery to selected regions of therespiratory system such as the deep lung or upper or central airways.For example, higher density or larger respirable dry particles may beused for upper airway delivery, or a mixture of varying size respirabledry particles in a sample, provided with the same or a differentformulation, may be administered to target different regions of the lungin one administration.

In order to relate the dispersion of powder at different inhalation flowrates, volumes, and from inhalers of different resistances, the energyrequired to perform the inhalation maneuver can be calculated.Inhalation energy can be calculated from the equation E=R²Q²V where E isthe inhalation energy in Joules, R is the inhaler resistance inkPa^(1/2)/LPM, Q is the steady flow rate in L/min and V is the inhaledair volume in L.

Healthy adult populations are predicted to be able to achieve inhalationenergies ranging from 2.9 to 22 Joules by using values of peakinspiratory flow rate (PIFR) measured by Clarke et al. (Journal ofAerosol Med, 6(2), p. 99-110, 1993) for the flow rate Q from two inhalerresistances of 0.02 and 0.055 kPa1/2/LPM, with a inhalation volume of 2L based on both FDA guidance documents for dry powder inhalers and onthe work of Tiddens et al. (Journal of Aerosol Med, 19, (4), p. 456-465,2006) who found adults averaging 2.2 L inhaled volume through a varietyof DPIs.

Mild, moderate and severe adult COPD patients are predicted to be ableto achieve inhalation energies of 5.1 to 21 Joules, 5.2 to 19 Joules,and 2.3 to 18 Joules respectively. This is again based on using measuredPIFR values for the flow rate Q in the equation for inhalation energy.The PIFR achievable for each group is a function of the inhalerresistance that is being inhaled through. The work of Broeders et al.(Eur Respir J, 18, p. 780-783, 2001) was used to predict maximum andminimum achievable PIFR through 2 dry powder inhalers of resistances0.021 and 0.032 kPa1/2/LPM for each.

Similarly, adult asthmatic patients are predicted to be able to achieveinhalation energies of 7.4 to 21 Joules based on the same assumptions asthe COPD population and PIFR data from Broeders et al.

Healthy adults, adult COPD patients, and asthmatic adults, for example,should be capable of providing sufficient inhalation energy to empty anddisperse the dry powder formulations of the invention. For example, a 25mg dose of Formulation III was found to require only 0.16 Joules toempty 80% of the fill weight in a single inhalation well deagglomeratedas illustrated by a Dv50 within 1 micrometer of that at much higherinhalation energies. All the adult patient populations listed above werecalculated to be able to achieve greater than 2 Joules, more than anorder of magnitude more inhalational energy than required.

An advantage of the invention is the production of powders that dispersewell across a wide range of flowrates and are relatively flowrateindependent. The dry particles and powders of the invention enable theuse of a simple, passive DPI for a wide patient population.

Methods

The respirable dry powders and respirable dry particles of the presentinvention are for administration to the respiratory tract. The drypowders and dry particles of the invention can be administered to asubject in need thereof for the treatment of respiratory (e.g.,pulmonary) diseases, such as asthma, airway hyperresponsiveness,seasonal allergic allergy, bronchiectasis, chronic bronchitis,emphysema, chronic obstructive pulmonary disease, cystic fibrosis andthe like, and for the treatment and/or prevention of acute exacerbationsof these chronic diseases, such as exacerbations caused by viralinfections (e.g., influenza virus, parainfluenza virus, respiratorysyncytial virus, rhinovirus, adenovirus, metapneumovirus, coxsackievirus, echo virus, corona virus, herpes virus, cytomegalovirus, and thelike), bacterial infections (e.g., Streptococcus pneumoniae, which iscommonly referred to as pneumococcus, Staphylococcus aureus,Burkholderis ssp., Streptococcus agalactiae, Haemophilus influenzae,Haemophilus parainfluenzae Klebsiella pneumoniae, Escherichia coli,Pseudomonas aeruginosa, Moraxella catarrhalis, Chlamydophila pneumoniae,Mycoplasma pneumoniae, Legionella pneumophila, Serratia marcescens,Mycobacterium tuberculosis, Bordetella pertussis, and the like), fungalinfections (e.g., Histoplasma capsulatum, Cryptococcus neoformans,Pneumocystis jiroveci, Coccidioides immitis, and the like) or parasiticinfections (e.g., Toxoplasma gondii, Strongyloides stercoralis, and thelike), or environmental allergens and irritants (e.g., aeroallergens,including pollen and cat dander, airborne particulates, and the like).

The dry powders and dry particles of the invention can be administeredto a subject in need thereof for the treatment and/or prevention and/orreducing contagion of infectious diseases of the respiratory tract, suchas pneumonia (including community-acquired pneumonia, nosocomialpneumonia (hospital-acquired pneumonia, HAP; health-care associatedpneumonia, HCAP), ventilator-associated pneumonia (VAP)),ventilator-associated tracheobronchitis (VAT), bronchitis, croup (e.g.,postintubation croup, and infectious croup), tuberculosis, influenza,common cold, and viral infections (e.g., influenza virus, parainfluenzavirus, respiratory syncytial virus, rhinovirus, adenovirus,metapneumovirus, coxsackie virus, echo virus, corona virus, herpesvirus, cytomegalovirus, and the like), bacterial infections (e.g.,Streptococcus pneumoniae, which is commonly referred to as pneumococcus,Staphylococcus aureus, Streptococcus agalactiae, Haemophilus influenzae,Haemophilus parainfluenzae Klebsiella pneumoniae, Escherichia coli,Pseudomonas aeruginosa, Moraxella catarrhalis, Chlamydophila pneumoniae,Mycoplasma pneumoniae, Legionella pneumophila, Serratia marcescens,Mycobacterium tuberculosis, Bordetella pertussis, and the like), fungalinfections (e.g., Histoplasma capsulatum, Cryptococcus neoformans,Pneumocystis jiroveci, Coccidioides immitis, and the like) or parasiticinfections (e.g., Toxoplasma gondii, Strongyloides stercoralis, and thelike), or environmental allergens and irritants (e.g., aeroallergens,airborne particulates, and the like).

The respirable dry particles and dry powder can be administered to alterthe biophysical and/or biological properties of the mucosal lining ofthe respiratory tract (e.g, the airway lining fluid) and underlyingtissue (e.g., respiratory tract epithelium). These properties include,for example, gelation at the mucus surface, surface tension of themucosal lining, surface elasticity and/or viscosity of the mucosallining, bulk elasticity and/or viscosity of the mucosal lining. Withoutwishing to be bound by a particular theory, it is believed that thebenefits produced by the respirable dry particles or dry powder and themethods described herein (e.g., therapeutic and prophylactic benefits),result from an increase in the amount of calcium cation (Ca²⁺ providedby the calcium salts in the respirable dry particles or dry powder) inthe respiratory tract (e.g., lung mucus or airway lining fluid) afteradministration of the respirable dry particles or dry powder.

The respirable dry powders and dry particles can be administered toincrease the rate of mucociliary clearance. Clearance of microbes andinhaled particles is an important function of airways to preventrespiratory infection and exposure to or systemic absorption ofpotentially noxious agents. This is performed as an integrated functionby epithelial, mucus-secreting, and immunologic response cells presentat the airway surface. It prominently includes the cilia at theepithelial cell airway surface, whose function is to beat synchronouslyto transport the overlying liquid mucus blanket proximally (toward themouth), where it exits the airway and is swallowed or expectorated.

The respirable dry powders and dry particles can be administered toassist in all of these functions. By increasing surface viscoelasticity,the respirable dry powders and dry particles retain microbes andparticulates at the surface of the airway mucus blanket, where they donot gain systemic exposure to the host. Hypertonic dry powders and dryparticles induce water/liquid transport out of the airway epithelialcells, making the peri-ciliary liquid layer less viscous and renderingciliary beating more effective in moving and clearing the overlyingmucus blanket. Dry particles and dry powders that contain calcium saltsas the pharmacologically active agent, also cause an increase in bothciliary beat frequency and the force or vigor of ciliary contractions,with resultant increase in clearance velocity of the overlying mucusstream.

Mucociliary clearance is measured by a well-established technique thatmeasures the function and speed of clearance quantitatively using safe,inhaled radioisotope preparation (e.g., Technitium (^(99m)Tc)) insolution. The radioisotope is measured quantitatively by externalscintigraphy. Serial measurements over several hours allow for theassessment of velocity of clearance and effect of a drug vs.baseline/control value.

In some aspects, the invention is a method for treating a pulmonarydiseases, such as asthma, airway hyperresponsiveness, seasonal allergicallergy, bronchiectasis, chronic bronchitis, emphysema, chronicobstructive pulmonary disease, cystic fibrosis and the like, comprisingadministering to the respiratory tract of a subject in need thereof aneffective amount of respirable dry particles or dry powder, as describedherein.

In other aspects, the invention is a method for the treatment orprevention of acute exacerbations of a chronic pulmonary disease, suchas asthma, airway hyperresponsiveness, seasonal allergic allergy,bronchiectasis, chronic bronchitis, emphysema, chronic obstructivepulmonary disease, cystic fibrosis and the like, comprisingadministering to the respiratory tract of a subject in need thereof aneffective amount of respirable dry particles or dry powder, as describedherein.

In other aspects, the invention is a method for treating, preventingand/or reducing contagion of an infectious disease of the respiratorytract, comprising administering to the respiratory tract of a subject inneed thereof an effective amount of respirable dry particles or drypowder, as described herein.

The respirable dry particles and dry powders can be administered to therespiratory tract of a subject in need thereof using any suitablemethod, such as instillation techniques, and/or an inhalation device,such as a dry powder inhaler (DPI) or metered dose inhaler (MDI). Anumber of DPIs are available, such as, the inhalers disclosed is U.S.Pat. Nos. 4,995,385 and 4,069,819, Spinhaler® (Fisons, Loughborough,U.K.), Rotahalers®, Diskhaler® and Diskus® (GlaxoSmithKline, ResearchTriangle Technology Park, North Carolina), FlowCapss® (Hovione, Loures,Portugal), Inhalators® (Boehringer-Ingelheim, Germany), Aerolizer®(Novartis, Switzerland), and others known to those skilled in the art.

Generally, inhalation devices (e.g., DPIs) are able to deliver a maximumamount of dry powder or dry particles in a single inhalation, which isrelated to the capacity of the blisters, capsules (e.g. size 000, 00,0E, 0, 1, 2, 3, and 4, with respective volumetric capacities of 1.37 ml,950 μl, 770 μl, 680 μl, 480 μl, 360 μl, 270 μl, and 200 μl) or othermeans that contain the dry particles or dry powders within the inhaler.Accordingly, delivery of a desired dose or effective amount may requiretwo or more inhalations. Preferably, each dose that is administered to asubject in need thereof contains an effective amount of respirable dryparticles or dry powder and is administered using no more than about 4inhalations. For example, each dose of respirable dry particles or drypowder can be administered in a single inhalation or 2, 3, or 4inhalations. The respirable dry particles and dry powders, arepreferably administered in a single, breath-activated step using abreath-activated DPI. When this type of device is used, the energy ofthe subject's inhalation both disperses the respirable dry particles anddraws them into the respiratory tract.

The respirable dry particles or dry powders can be delivered byinhalation to a desired area within the respiratory tract, as desired.It is well-known that particles with an aerodynamic diameter of about 1micron to about 3 microns, can be delivered to the deep lung. Largeraerodynamic diameters, for example, from about 3 microns to about 5microns can be delivered to the central and upper airways.

It is believed that when some dry powders that contain divalent metalsalts as active ingredients are administered, there is a possibilitythat at least some of the respirable dry powder will deposit in the oralcavity and produce an unpleasant “salty mouth” sensation. It isenvisioned that this sensation could lead patients to not comply withtherapeutic instructions or to discontinue therapy. An advantage of therespirable dry powders of this invention is that they are small andhighly dispersible, and therefore, deposition in the oral cavity isreduced and the occurrence of an unpleasant salty mouth sensation isreduced or prevented.

For dry powder inhalers, oral cavity deposition is dominated by inertialimpaction and so characterized by the aerosol's Stokes number (DeHaan etal. Journal of Aerosol Science, 35 (3), 309-331, 2003). For equivalentinhaler geometry, breathing pattern and oral cavity geometry, the Stokesnumber, and so the oral cavity deposition, is primarily affected by theaerodynamic size of the inhaled powder. Hence, factors which contributeto oral deposition of a powder include the size distribution of theindividual particles and the dispersibility of the powder. If the MMADof the individual particles is too large, e.g. above 5 μm, then anincreasing percentage of powder will deposit in the oral cavity.Likewise, if a powder has poor dispersibility, it is an indication thatthe particles will leave the dry powder inhaler and enter the oralcavity as agglomerates. Agglomerated powder will perform aerodynamicallylike an individual particle as large as the agglomerate, therefore evenif the individual particles are small (e.g., MMAD of 5 microns or less),the size distribution of the inhaled powder may have an MMAD of greaterthan 5 μm, leading to enhanced oral cavity deposition.

Therefore, it is desirable to have a powder in which the particles aresmall (e.g., MMAD of 5 microns or less, e.g. between 1 to 5 microns),and are highly dispersible (e.g. 1/4 bar or alternatively, 0.5/4 bar of2.0, and preferably less than 1.5). More preferably, the respirable drypowder is comprised of respirable dry particles with an MMAD between 1to 4 microns or 1 to 3 microns, and have a 1/4 bar less than 1.4, orless than 1.3, and more preferably less than 1.2.

The absolute geometric diameter of the particles measured at 1 bar usingthe HELOS system is not critical provided that the particle's envelopedensity is sufficient such that the MMAD is in one of the ranges listedabove, wherein MMAD is VMGD times the square root of the envelopedensity (MMAD=VMGD*sqrt(envelope density)). If it is desired to delivera high unit dose of salt using a fixed volume dosing container, then,particles of higher envelop density are desired. High envelope densityallows for more mass of powder to be contained within the fixed volumedosing container. Preferable envelope densities are greater than 0.1g/cc, greater than 0.25 g/cc, greater than 0.4 g/cc, greater than 0.5g/cc, and greater than 0.6 g/cc.

The respirable dry powders and particles of the invention can beemployed in compositions suitable for drug delivery via the respiratorysystem. For example, such compositions can include blends of therespirable dry particles of the invention and one or more other dryparticles or powders, such as dry particles or powders that containanother active agent, or that consist of or consist essentially of oneor more pharmaceutically acceptable excipients.

Respirable dry powders and dry particles suitable for use in the methodsof the invention can travel through the upper airways (i.e., theoropharynx and larynx), the lower airways, which include the tracheafollowed by bifurcations into the bronchi and bronchioli, and throughthe terminal bronchioli which in turn divide into respiratory bronchiolileading then to the ultimate respiratory zone, the alveoli or the deeplung. In one embodiment of the invention, most of the mass of respirabledry powders or particles deposit in the deep lung. In another embodimentof the invention, delivery is primarily to the central airways. Inanother embodiment, delivery is to the upper airways.

The respirable dry particles or dry powders of the invention can bedelivered by inhalation at various parts of the breathing cycle (e.g.,laminar flow at mid-breath). An advantage of the high dispersibility ofthe dry powders and dry particles of the invention is the ability totarget deposition in the respiratory tract. For example, breathcontrolled delivery of nebulized solutions is a recent development inliquid aerosol delivery (Dalby et al. in Inhalation Aerosols, edited byHickey 2007, p. 437). In this case, nebulized droplets are released onlyduring certain portions of the breathing cycle. For deep lung delivery,droplets are released in the beginning of the inhalation cycle, whilefor central airway deposition, they are released later in theinhalation.

The highly dispersible powders of this invention provide advantages fortargeting the timing of drug delivery in the breathing cycle and alsolocation in the human lung. Because the respirable dry powders of theinvention can be dispersed rapidly, such as within a fraction of atypical inhalation maneuver, the timing of the powder dispersal can becontrolled to deliver an aerosol at specific times within theinhalation.

With a highly dispersible powder, the complete dose of aerosol can bedispersed at the beginning portion of the inhalation. While thepatient's inhalation flow rate ramps up to the peak inspiratory flowrate, a highly dispersible powder will begin to disperse already at thebeginning of the ramp up and could completely disperse a dose in thefirst portion of the inhalation. Since the air that is inhaled at thebeginning of the inhalation will ventilate deepest into the lungs,dispersing the most aerosol into the first part of the inhalation ispreferable for deep lung deposition. Similarly, for central deposition,dispersing the aerosol at a high concentration into the air which willventilate the central airways can be achieved by rapid dispersion of thedose near the mid to end of the inhalation. This can be accomplished bya number of mechanical and other means such as a switch operated bytime, pressure or flow rate which diverts the patient's inhaled air tothe powder to be dispersed only after the switch conditions are met.

Aerosol dosage, formulations and delivery systems may be selected for aparticular therapeutic application, as described, for example, in Gonda,I. “Aerosols for delivery of therapeutic and diagnostic agents to therespiratory tract,” in Critical Reviews in Therapeutic Drug CarrierSystems, 6: 273-313 (1990); and in Moren, “Aerosol Dosage Forms andFormulations,” in Aerosols in Medicine, Principles, Diagnosis andTherapy, Moren, et al., Eds., Elsevier, Amsterdam (1985).

As described herein, it is believed that the therapeutic andprophylactic effects of the respirable dry particles and dry powders arethe result of an increased amount of calcium in the respiratory tract(e.g., lung) following administration of respirable dry particles anddry powders. Accordingly, since the amount of calcium provided can varydepending upon the particular salt selected, dosing can be based on thedesired amount of calcium to be delivered to the lung. For example, onemole of calcium chloride (CaCl₂) dissociates to provide one mole ofCa²⁺, but one mole of calcium citrate can provide three moles of Ca²⁺.

Generally, an effective amount of a pharmaceutical formulation willdeliver a dose of about 0.001 mg Ca⁺²/kg body weight/dose to about 2 mgCa⁺²/kg body weight/dose, about 0.002 mg Ca⁺²/kg body weight/dose toabout 2 mg Ca⁺²/kg body weight/dose, about 0.005 mg Ca⁺²/kg bodyweight/dose to about 2 mg Ca⁺²/kg body weight/dose, about 0.01 mgCa⁺²/kg body weight/dose to about 2 mg Ca⁺²/kg body weight/dose, about0.01 mg Ca⁺²/kg body weight/dose to about 60 mg Ca⁺²/kg bodyweight/dose, about 0.01 mg Ca⁺²/kg body weight/dose to about 50 mgCa⁺²/kg body weight/dose, about 0.01 mg Ca⁺²/kg body weight/dose toabout 40 mg Ca⁺²/kg body weight/dose, about 0.01 mg Ca⁺²/kg bodyweight/dose to about 30 mg Ca⁺²/kg body weight/dose, about 0.01 mgCa⁺²/kg body weight/dose to about 20 mg Ca⁺²/kg body weight/dose, about0.01 mg Ca⁺²/kg body weight/dose to about 10 mg Ca⁺²/kg bodyweight/dose, about 0.01 mg Ca⁺²/kg body weight/dose to about 5 mgCa⁺²/kg body weight/dose, about 0.01 mg Ca⁺²/kg body weight/dose toabout 2 mg Ca⁺²/kg body weight/dose, about 0.02 mg Ca⁺²/kg bodyweight/dose to about 2 mg Ca⁺²/kg body weight/dose, about 0.03 mgCa⁺²/kg body weight/dose to about 2 mg Ca⁺²/kg body weight/dose, about0.04 mg Ca⁺²/kg body weight/dose to about 2 mg Ca⁺²/kg body weight/dose,about 0.05 mg Ca⁺²/kg body weight/dose to about 2 mg Ca⁺²/kg bodyweight/dose, about 0.1 mg Ca⁺²/kg body weight/dose to about 2 mg Ca⁺²/kgbody weight/dose, about 0.1 mg Ca⁺²/kg body weight/dose to about 1 mgCa⁺²/kg body weight/dose, about 0.1 mg Ca⁺²/kg body weight/dose to about0.5 mg Ca⁺²/kg body weight/dose, about 0.2 mg Ca⁺²/kg body weight/doseto about 0.5 mg Ca⁺²/kg body weight/dose, about 0.18 mg Ca⁺²/kg bodyweight/dose, about 0.001 mg Ca⁺²/kg body weight/dose, about 0.005 mgCa⁺²/kg body weight/dose, about 0.01 mg Ca⁺²/kg body weight/dose, about0.02 mg Ca⁺²/kg body weight/dose, or about 0.5 mg Ca⁺²/kg bodyweight/dose.

In some embodiments the amount of calcium delivered to the respiratorytract (e.g., lungs, respiratory airway) is about 0.001 mg Ca⁺²/kg bodyweight/dose to about 2 mg Ca⁺²/kg body weight/dose, about 0.002 mgCa⁺²/kg body weight/dose to about 2 mg Ca⁺²/kg body weight/dose, about0.005 mg Ca⁺²/kg body weight/dose to about 2 mg Ca⁺²/kg bodyweight/dose, about 0.01 mg Ca⁺²/kg body weight/dose to about 2 mgCa⁺²/kg body weight/dose, about 0.01 mg Ca⁺²/kg body weight/dose toabout 60 mg Ca⁺²/kg body weight/dose, about 0.01 mg Ca⁺²/kg bodyweight/dose to about 50 mg Ca⁺²/kg body weight/dose, about 0.01 mgCa⁺²/kg body weight/dose to about 40 mg Ca⁺²/kg body weight/dose, about0.01 mg Ca⁺²/kg body weight/dose to about 30 mg Ca⁺²/kg bodyweight/dose, about 0.01 mg Ca⁺²/kg body weight/dose to about 20 mgCa⁺²/kg body weight/dose, about 0.01 mg Ca⁺²/kg body weight/dose toabout 10 mg Ca⁺²/kg body weight/dose, about 0.01 mg Ca⁺²/kg bodyweight/dose to about 5 mg Ca⁺²/kg body weight/dose, about 0.01 mgCa⁺²/kg body weight/dose to about 2 mg Ca⁺²/kg body weight/dose, about0.02 mg Ca⁺²/kg body weight/dose to about 2 mg Ca⁺²/kg body weight/dose,about 0.03 mg Ca⁺²/kg body weight/dose to about 2 mg Ca⁺²/kg bodyweight/dose, about 0.04 mg Ca⁺²/kg body weight/dose to about 2 mgCa⁺²/kg body weight/dose, about 0.05 mg Ca⁺²/kg body weight/dose toabout 2 mg Ca⁺²/kg body weight/dose, about 0.1 mg Ca⁺²/kg bodyweight/dose to about 2 mg Ca⁺²/kg body weight/dose, about 0.1 mg Ca⁺²/kgbody weight/dose to about 1 mg Ca⁺²/kg body weight/dose, about 0.1 mgCa⁺²/kg body weight/dose to about 0.5 mg Ca⁺²/kg body weight/dose, about0.2 mg Ca⁺²/kg body weight/dose to about 0.5 mg Ca⁺²/kg bodyweight/dose, about 0.18 mg Ca⁺²/kg body weight/dose, about 0.001 mgCa⁺²/kg body weight/dose, about 0.005 mg Ca⁺²/kg body weight/dose, about0.01 mg Ca⁺²/kg body weight/dose, about 0.02 mg Ca⁺²/kg bodyweight/dose, or about 0.5 mg Ca⁺²/kg body weight/dose.

In other embodiments the amount of calcium delivered to the upperrespiratory tract (e.g., nasal cavity) is of about 0.001 mg Ca⁺²/kg bodyweight/dose to about 2 mg Ca⁺²/kg body weight/dose, about 0.002 mgCa⁺²/kg body weight/dose to about 2 mg Ca⁺²/kg body weight/dose, about0.005 mg Ca⁺²/kg body weight/dose to about 2 mg Ca⁺²/kg bodyweight/dose, about 0.01 mg Ca⁺²/kg body weight/dose to about 2 mgCa⁺²/kg body weight/dose, about 0.01 mg Ca⁺²/kg body weight/dose toabout 60 mg Ca⁺²/kg body weight/dose, about 0.01 mg Ca⁺²/kg bodyweight/dose to about 50 mg Ca⁺²/kg body weight/dose, about 0.01 mgCa⁺²/kg body weight/dose to about 40 mg Ca⁺²/kg body weight/dose, about0.01 mg Ca⁺²/kg body weight/dose to about 30 mg Ca⁺²/kg bodyweight/dose, about 0.01 mg Ca⁺²/kg body weight/dose to about 20 mgCa⁺²/kg body weight/dose, about 0.01 mg Ca⁺²/kg body weight/dose toabout 10 mg Ca⁺²/kg body weight/dose, about 0.01 mg Ca⁺²/kg bodyweight/dose to about 5 mg Ca⁺²/kg body weight/dose, about 0.01 mgCa⁺²/kg body weight/dose to about 2 mg Ca⁺²/kg body weight/dose, about0.02 mg Ca⁺²/kg body weight/dose to about 2 mg Ca⁺²/kg body weight/dose,about 0.03 mg Ca⁺²/kg body weight/dose to about 2 mg Ca⁺²/kg bodyweight/dose, about 0.04 mg Ca⁺²/kg body weight/dose to about 2 mgCa⁺²/kg body weight/dose, about 0.05 mg Ca⁺²/kg body weight/dose toabout 2 mg Ca⁺²/kg body weight/dose, about 0.1 mg Ca⁺²/kg bodyweight/dose to about 2 mg Ca⁺²/kg body weight/dose, about 0.1 mg Ca⁺²/kgbody weight/dose to about 1 mg Ca⁺²/kg body weight/dose, about 0.1 mgCa⁺²/kg body weight/dose to about 0.5 mg Ca⁺²/kg body weight/dose, about0.2 mg Ca⁺²/kg body weight/dose to about 0.5 mg Ca⁺²/kg bodyweight/dose, about 0.18 mg Ca⁺²/kg body weight/dose, about 0.001 mgCa⁺²/kg body

In addition, when the respirable dry particles and dry powders include asodium salt, the respirable dry particles and dry powders can beadministered in an amount sufficient to deliver a dose of about 0.001 mgNa⁺/kg body weight/dose to about 10 mg Na⁺/kg body weight/dose, or about0.01 mg Na⁺/kg body weight/dose to about 10 mg Na⁺/kg body weight/dose,or about 0.1 mg Na⁺/kg body weight/dose to about 10 mg Na⁺/kg bodyweight/dose, or about 1.0 mg Na⁺/kg body weight/dose to about 10 mgNa⁺/kg body weight/dose, or about 0.001 mg Na⁺/kg body weight/dose toabout 1 mg Na⁺/kg body weight/dose, or about 0.01 mg Na⁺/kg bodyweight/dose to about 1 mg Na⁺/kg body weight/dose, or about 0.1 mgNa⁺/kg body weight/dose to about 1 mg Na⁺/kg body weight/dose, about 0.2to about 0.8 mg Na⁺/kg body weight/dose, about 0.3 to about 0.7 mgNa⁺/kg body weight/dose, or about 0.4 to about 0.6 mg Na⁺/kg bodyweight/dose.

In some embodiments the amount of sodium delivered to the respiratorytract (e.g., lungs, respiratory airway) is about 0.001 mg/kg bodyweight/dose to about 10 mg/kg body weight/dose, or about 0.01 mg/kg bodyweight/dose to about 10 mg/kg body weight/dose, or about 0.1 mg/kg bodyweight/dose to about 10 mg/kg body weight/dose, or about 1 mg/kg bodyweight/dose to about 10 mg/kg body weight/dose, or about 0.001 mg/kgbody weight/dose to about 1 mg/kg body weight/dose, or about 0.01 mg/kgbody weight/dose to about 1 mg/kg body weight/dose, or about 0.1 mg/kgbody weight/dose to about 1 mg/kg body weight/dose, or about 0.2 toabout 0.8 mg/kg body weight/dose, or about 0.3 to about 0.7 mg/kg bodyweight/dose, or about 0.4 to about 0.6 mg/kg body weight/dose.

In other embodiments the amount of sodium delivered to the upperrespiratory tract (e.g., nasal cavity) is about 0.001 mg/kg bodyweight/dose to about 10 mg/kg body weight/dose, or about 0.01 mg/kg bodyweight/dose to about 10 mg/kg body weight/dose, or about 0.1 mg/kg bodyweight/dose to about 10 mg/kg body weight/dose, or about 1 mg/kg bodyweight/dose to about 10 mg/kg body weight/dose, or about 0.001 mg/kgbody weight/dose to about 1 mg/kg body weight/dose, or about 0.01 mg/kgbody weight/dose to about 1 mg/kg body weight/dose, or about 0.1 mg/kgbody weight/dose to about 1 mg/kg body weight/dose, or about 0.2 toabout 0.8 mg/kg body weight/dose, or about 0.3 to about 0.7 mg/kg bodyweight/dose, or about 0.4 to about 0.6 mg/kg body weight/dose.

Suitable intervals between doses that provide the desired therapeuticeffect can be determined based on the severity of the condition (e.g.,infection), overall well being of the subject and the subject'stolerance to respirable dry particles and dry powders and otherconsiderations. Based on these and other considerations, a clinician candetermine appropriate intervals between doses. Generally, respirable dryparticles and dry powders are administered once, twice or three times aday, as needed.

If desired or indicated, the respirable dry particles and dry powdersdescribed herein can be administered with one or more other therapeuticagents. The other therapeutic agents can be administered by any suitableroute, such as orally, parenterally (e.g., intravenous, intraarterial,intramuscular, or subcutaneous injection), topically, by inhalation(e.g., intrabronchial, intranasal or oral inhalation, intranasal drops),rectally, vaginally, and the like. The respirable dry particles and drypowders can be administered before, substantially concurrently with, orsubsequent to administration of the other therapeutic agent. Preferably,the respirable dry particles and dry powders and the other therapeuticagent are administered so as to provide substantial overlap of theirpharmacologic activities.

Another advantage provided by the respirable dry powders and respirabledry particles described herein, is that dosing efficiency can beincreased as a result of hygroscopic growth of particles inside thelungs, due to particle moisture growth. The propensity of the partiallyamorphous, high salt compositions of the invention to take up water atelevated humidities can also be advantageous with respect to theirdeposition profiles in vivo. Due to their rapid water uptake at highhumidities, these powder formulations can undergo hygroscopic growth dothe absorbance of water from the humid air in the respiratory tract asthey transit into the lungs. This can result in an increase in theireffective aerodynamic diameters during transit into the lungs, whichwill further facilitate their deposition in the airways.

EXEMPLIFICATION

Materials used in the following Examples and their sources are listedbelow. Calcium chloride dihydrate, calcium lactate pentahydrate, sodiumchloride, L-leucine, maltodextrin, mannitol, lactose and trehalose wereobtained from Sigma-Aldrich Co. (St. Louis, Mo.) or Spectrum Chemicals(Gardena, Calif.); sodium sulfate from EMD Chemicals (Gibbstown, N.J.),Sigma-Aldrich Co. (St. Louis, Mo.) or Spectrum Chemicals (Gardena,Calif.); and sodium citrate dihydrate from J.T. Baker (Phillipsburg,N.J.), Mallinckrodt Baker (Phillipsburg, N.J.) or Spectrum Chemicals(Gardena, Calif.). Ultrapure water was from a water purification system(Millipore Corp., Billerica, Mass.).

Methods:

Geometric or Volume Diameter.

Volume median diameter (×50), which may also be referred to as volumemedian geometric diameter (VMGD), was determined using a laserdiffraction technique. The equipment consisted of a HELOS diffractometerand a RODOS dry powder disperser (Sympatec, Inc., Princeton, N.J.). TheRODOS disperser applies a shear force to a sample of particles,controlled by the regulator pressure (typically set at 1.0 bar withorifice ring pressure set at 7 mbar) of the incoming compressed dry air.The pressure settings may be varied to vary the amount of energy used todisperse the powder. For example, the regulator pressure may be variedfrom 0.2 bar to 4.0 bar; and the orifice ring pressure may be variedfrom 5.00 mbar to 115.00 mbar. Powder sample is dispensed from amicrospatula into the RODOS funnel. The dispersed particles travelthrough a laser beam where the resulting diffracted light patternproduced is collected, typically using an R2 lens, by a series ofdetectors. The ensemble diffraction pattern is then translated into avolume-based particle size distribution using the Fraunhofer diffractionmodel, on the basis that smaller particles diffract light at largerangles. Using this method geometric standard deviation (GSD) for thevolume mean geometric diameter was also determined.

Fine Particle Fraction.

The aerodynamic properties of the powders dispersed from an inhalerdevice were assessed with a Mk-II 1 ACFM Andersen Cascade Impactor(Copley Scientific Limited, Nottingham, UK). The instrument was run incontrolled environmental conditions of 18 to 25° C. and relativehumidity (RH) between 20 and 40%. The instrument consists of eightstages that separate aerosol particles based on inertial impaction. Ateach stage, the aerosol stream passes through a set of nozzles andimpinges on a corresponding impaction plate. Particles having smallenough inertia will continue with the aerosol stream to the next stage,while the remaining particles will impact upon the plate. At eachsuccessive stage, the aerosol passes through nozzles at a highervelocity and aerodynamically smaller particles are collected on theplate. After the aerosol passes through the final stage, a filtercollects the smallest particles that remain. Gravimetric and/or chemicalanalyses can then be performed to determine the particle sizedistribution. A short stack cascade impactor is also utilized to allowfor reduced labor time to evaluate two aerodynamic particle sizecut-points. With this collapsed cascade impactor, stages are eliminatedexcept those required to establish fine and coarse particle fractions.

The impaction techniques utilized allowed for the collection of two oreight separate powder fractions. The capsules (HPMC, Size 3; ShionogiQualicaps, Madrid, Spain) were approximately half-filled with powder andplaced in a hand-held, breath-activated dry powder inhaler (DPI) device,the high resistance RS-01 DPI (Plastiape, Osnago, Italy). The capsulewas punctured and the powder was drawn through the cascade impactoroperated at a flow rate of 60.0 L/min for 2.0 s. At this flowrate, thecalibrated cut-off diameters for the eight stages are 8.6, 6.5, 4.4,3.3, 2.0, 1.1, 0.5 and 0.3 microns and for the two stages used with theshort stack cascade impactor, the cut-off diameters are 5.6 microns and3.4 microns. The fractions were collected by placing filters in theapparatus and determining the amount of powder that impinged on them bygravimetric measurements or chemical measurements on an HPLC, as labeledin the tables. The fine particle fraction of the total dose of powder(FPF_TD) less than or equal to an effective cut-off aerodynamic diameterwas calculated by dividing the powder mass recovered from the desiredstages of the impactor by the total particle mass in the capsule.Results are reported as the fine particle fraction of less than 5.6microns (FPF <5.6 microns) and the fine particle fraction of less than3.4 microns (FPF <3.4 microns). The fine particle fraction canalternatively be calculated relative to the recovered or emitted dose ofpowder by dividing the powder mass recovered from the desired stages ofthe impactor by the total powder mass recovered.

Aerodynamic Diameter.

Mass median aerodynamic diameter (MMAD) was determined using theinformation obtained by the Andersen Cascade Impactor. The cumulativemass under the stage cut-off diameter is calculated for each stage andnormalized by the recovered dose of powder. The MMAD of the powder isthen calculated by linear interpolation of the stage cut-off diametersthat bracket the 50th percentile.

Emitted Dose.

A measure of the emission properties of the powders was determined byusing the information obtained from the Andersen Cascade Impactor tests.The filled capsule weight was recorded at the beginning of the run andthe final capsule weight was recorded after the completion of the run.The difference in weight represented the amount of powder emitted fromthe capsule (CEPM or capsule emitted powder mass). The emitted dose wascalculated by dividing the amount of powder emitted from the capsule bythe total initial particle mass in the capsule.

Tap Density.

Two methods were utilized to measure tap density. (1) A modified methodrequiring smaller powder quantities was initially used, following USP<616> with the substitution of a 1.5 cc microcentrifuge tube (EppendorfAG, Hamburg, Germany) to hold the powder. (2) USP <616> was used,utilizing a 100 cc graduated cylinder. Instruments for measuring tapdensity, known to those skilled in the art, include but are not limitedto the Dual Platform Microprocessor Controlled Tap Density Tester(Vankel, Cary, N.C.) or a GeoPyc instrument (Micrometrics InstrumentCorp., Norcross, Ga.). Tap density is a standard measure of the envelopemass density. The envelope mass density of an isotropic particle isdefined as the mass of the particle divided by the minimum sphericalenvelope volume within which it can be enclosed.

Scanning Electron Microscopy (SEM).

SEM was performed using a FEI Quanta 200 scanning electron microscope(Hillsboro, Oreg.) equipped with an Everhart Thornley (ET) detector.Images were collected and analysed using xTm (v. 2.01) and XT Docu (v.3.2) software, respectively. The magnification was verified using a NISTtraceable standard. Each sample was prepared for analysis by placing asmall amount on a carbon adhesive tab supported on an aluminum mount.Each sample was then sputter coated with Au/Pd using a Cressington 108auto Sputter Coater at approximately 20 mA and 0.13 mbar (Ar) for 75seconds. The data acquisition parameters are displayed in theinformation bar at the bottom of each image. The magnification reportedon each image was calculated upon the initial data acquisition. Thescale bar reported in the lower portion of each image is accurate uponresizing and should be used when making size determinations.

Liquid Feedstock Preparation for Spray Drying.

Spray drying homogenous particles requires that the ingredients ofinterest be solubilized in solution or suspended in a uniform and stablesuspension. Certain calcium salts, such as calcium chloride, calciumacetate and calcium lactate, are sufficiently water-soluble to preparesuitable spray drying solutions. However, other calcium salts, such ascalcium sulfate, calcium citrate and calcium carbonate, have a lowsolubility in water. The solubility in water of exemplary calcium saltsare listed in Table 1. As a result of these low solubilities,formulation feedstock development work was necessary to preparesolutions or suspensions that could be spray dried. These solutions orsuspensions included combinations of salts in an appropriate solvent,typically water but also ethanol and water mixtures or other solvents asdescribed earlier in the specification.

TABLE 1 Calcium Salts' Solubility in Water Calcium Salt Solubility inWater (at 20-30° C., 1 bar) Salt Water solubility (g/L) Calcium chloride1368^(1,2) Calcium acetate 347¹ Calcium lactate 105¹ Calcium gluconate   33.23³ Calcium sulfate    2.98¹ Calcium citrate    0.96¹ Calciumphosphate dibasic    0.2¹ Calcium carbonate Pract. Insol.² Calciumstearate Pract. Insol.² Calcium alginate Not applicable Sodium Carbonate505¹ Sodium Chloride 360¹ Sodium Citrate 910¹ Sodium Sulfate 194¹¹Perry, Robert H., Don W. Green, and James O. Maloney. Perry's ChemicalEngineers' Handbook. 7th ed. New York: McGraw-Hill, 1997. Print.²Solubility at 60° C. ³O'Neil, Maryadele J. The Merck Index: anEncyclopedia of Chemicals, Drugs, and Biologicals. 14th ed. WhitehouseStation, N.J.: Merck, 2006. Print.

As mentioned previously, calcium chloride has high water solubility.Sodium salts, such as sodium sulfate, sodium citrate and sodiumcarbonate, are also very soluble in water. As will be discussed furtherin the following examples, calcium chloride and sodium salts (the“starting materials”) are combined in solution or suspension to obtainstable calcium salts in final dry powder form. When combining thecalcium chloride and sodium salt in solution, the calcium and the anioncontributed from the sodium salt may react in a precipitation reactionto produce the desired calcium salt (i.e., CaCl₂+2NaXX→CaXX+2NaCl). Inthis case, the maximum solids concentration that maintained a clearsolution or a stable suspension were used for spray drying. Certaincalcium salts were soluble enough to be dissolved in water and thenspray dried alone. The same concept may be applied to, for example,magnesium salts by using magnesium chloride, potassium salts usingpotassium chloride, and sodium salts.

The starting materials may be provided in molar amounts where the fullprecipitation reaction may proceed to completion, termed ‘reaction tocompletion.’ The weight percent of calcium ion in exemplary calciumsalts are further listed in Table 2.

TABLE 2 Weight Percent of Ca²⁺ in Salt Molecules Weight % of Calcium ionin Salt Molecule Weight % of Ca²⁺ in Salt Formula MW molecule Calciumcarbonate CaCO₃ 100.09 40.0 Calcium chloride CaCl₂ 110.98 36.0 Calciumphosphate dibasic CaHPO₄ 136.06 29.4 Calcium sulfate CaSO₄ 136.14 29.4Calcium acetate Ca(C₂H₃O₂)₂ 158.17 25.3 Calcium citrate Ca₃(C₆H₅O₇)₂498.46 24.1 Calcium lactate Ca(C₃H₅O₃)₂ 218.218 18.3 Calcium sorbateCaC₁₂H₁₄O₄ 262.33 15.2 Calcium gluconate CaC₁₂H₂₂O₁₄ 430.373 9.3 Calciumstearate CaC₃₆H₇₀O₄ 607.02 6.6 Calcium alginate [Ca(C₆H₇O₆)₂]_(n) NA NA

Alternatively, excess calcium chloride may be added for an incompletereaction, or ‘reaction not to completion,’ where a given amount ofcalcium chloride is present in the final powder form. While calciumchloride is hygroscopic, its high water solubility may be beneficial tohave in small amounts in the final product to increase the solubility ofthe final product, to be able to tailor the dissolution profile, and toincrease the relative calcium ion ratio to sodium or other cationspresent in the formulation. For ease of formulation development, therequired molar ratios of calcium chloride and sodium salt were convertedto mass ratios of calcium chloride and sodium salt. An example is forcalcium citrate (i.e., calcium chloride+sodium citrate), where theprecipitation reaction proceeds forward as follows:

3CaCl₂+2Na₃C₆H₅O₇→Ca₃(C₆H₅O₇)₂+6NaCl

This reaction results in a 1:2 molar ratio of Ca:Na ions. For thereaction to proceed to completion, 3 moles of calcium chloride and 2moles of sodium citrate are required. To convert to mass in grams and aweight ratio, the moles of salts are multiplied by the molecular weightof the salts in grams per mole:

For calcium chloride: 3 mol CaCl₂×111 g/mol=333 g CaCl₂

For sodium citrate: 2 mol Na₃C₆H₅O₇×258 g/mol=516 g Na₃C₆H₅O₇

Therefore, a 1:1.55 or 39:61 weight ratio of CaCl₂:Na₃C₆H₅O₇ is requiredfor a complete reaction. These ratios were solubilized and spray driedto produce ‘pure salt’ formulations. In addition, dry powders wereproduced with an additional excipient, such as leucine or lactose. Theratio of calcium to sodium salt remained the same so as to produce a‘reaction to completion.’ For example, for a formulation of 50% (w/w)leucine, the remainder is composed of salts, such as calcium citrate(i.e., CaCl₂:Na₃C₆H₅O₇) where the 39:61, CaCl₂:Na₃C₆H₅O₇ weight ratio ismaintained. Thus, for that reaction: 50% (w/w) leucine, 19.5% (w/w)CaCl₂ and 30.5% (w/w) Na₃C₆H₅O₇ will be added. For a spray dryingprocess, the salts and other excipients will be dissolved or suspendedin a solvent (i.e., water). The solids concentration (w/v) can be chosendepending on the solubility of the different components. For the citrateformulation, a concentration of 5 mg/mL was appropriate, given thelimited solubility of calcium citrate: 0.95 mg/mL. Therefore, 5 g ofsolids (i.e., 2.5 g leucine, 0.975 g calcium chloride and 1.525 g ofsodium citrate) were dissolved in 1 L of ultrapure water.

In addition, when preparing spray drying solutions, the water weight ofthe hydrated starting material must be accounted for. The ratios usedfor formulations were based on the molecular weight of the anhydroussalts. For certain salts, hydrated forms are more readily available thanthe anhydrous form. This required an adjustment in the ratios originallycalculated, using a multiplier to correlate the molecular weight of theanhydrous salt with the molecular weight of the hydrate. An example ofthis calculation is included below.

For the example above, calcium chloride anhydrous molecular weight is110.98 g/mol and the dihydrate molecular weight is 147.01 g/mol. Sodiumcitrate anhydrous molecular weight is 258.07 g/mol and the dihydratemolecular weight is 294.10 g/mol.

The multiplier is analogous to the ratio of the dihydrate to anhydrousmolecular weight, e.g., 1.32 for calcium chloride and 1.14 for sodiumcitrate. Therefore, adjusting for the dihydrate forms results in: 2.5 gleucine, 1.287 g (i.e., 0.975 g×1.32) calcium chloride dihydrate and1.738 g (i.e., 1.525 g×1.14) of sodium citrate dihydrate were dissolvedand spray dried.

Spray Drying Using Niro Spray Dryer.

Dry powders were produced by spray drying utilizing a Niro Mobile Minorspray dryer (GEA Process Engineering Inc., Columbia, Md.) with powdercollection from a cyclone, a product filter or both. Atomization of theliquid feed was performed using a co-current two-fluid nozzle eitherfrom Niro (GEA Process Engineering Inc., Columbia, Md.) or a SprayingSystems (Carol Stream, Ill.) two-fluid nozzle with gas cap 67147 andfluid cap 2850SS, although other two-fluid nozzle setups are alsopossible. Additional atomization techniques include rotary atomizationor a pressure nozzle. The liquid feed was fed using gear pumps(Cole-Panner Instrument Company, Vernon Hills, Ill.) directly into thetwo-fluid nozzle or into a static mixer (Charles Ross & Son Company,Hauppauge, N.Y.) immediately before introduction into the two-fluidnozzle. An additional liquid feed technique includes feeding from apressurized vessel. Nitrogen or air may be used as the drying gas,provided that moisture in the air is at least partially removed beforeits use. Pressurized nitrogen or air can be used as the atomization gasfeed to the two-fluid nozzle. The process gas inlet temperature canrange from 100° C. to 300° C. and outlet temperature from 50° C. to 120°C. with a liquid feedstock rate of 20 mL/min to 100 mL/min. The gassupplying the two-fluid atomizer can vary depending on nozzle selectionand for the Niro co-current two-fluid nozzle can range from 8 kg/hr to15 kg/hr and be set a pressures ranging from 0.5 bar to 2.0 bar or forthe Spraying Systems two-fluid nozzle with gas cap 67147 and fluid cap2850SS can range from 40 to 100 g/min The atomizing gas rate can be setto achieve a certain gas to liquid mass ratio, which directly affectsthe droplet size created. The pressure inside the drying drum can rangefrom +3″ WC to −6″ WC. Spray dried powders can be collected in acontainer at the outlet of the cyclone, onto a cartridge or baghousefilter, or from both a cyclone and a cartridge or baghouse filter.

Spray Drying Using Büchi Spray Dryer. Dry powders were prepared by spraydrying on a Büchi B-290 Mini Spray Dryer (BÜCHI Labortechnik AG, Flawil,Switzerland) with powder collection from either a standard or HighPerformance cyclone. The system used the Büchi B-296 dehumidifier toensure stable temperature and humidity of the air used to spray dry.Furthermore, when the relative humidity in the room exceeded 30% RH, anexternal LG dehumidifier (model 49007903, LG Electronics, EnglewoodCliffs, N.J.) was run constantly. Atomization of the liquid feedutilized a Büchi two-fluid nozzle with a 1.5 mm diameter. Inlettemperature of the process gas can range from 100° C. to 220° C. andoutlet temperature from 80° C. to 120° C. with a liquid feedstockflowrate of 3 mL/min to 10 mL/min. The two-fluid atomizing gas rangesfrom 25 mm to 45 mm (300 LPH to 530 LPH) and the aspirator rate from 70%to 100% (28 m³/hr to 38 m³/hr).

Table 3 provides feedstock formulations used in preparation of some drypowders described herein.

TABLE 3 Feedstock Formulations Formulation Composition (w/w) I 10.0%leucine, 35.1% calcium chloride, 54.9% sodium citrate II 10.0% leucine,58.6% calcium lactate, 31.4% sodium chloride III 10.0% leucine, 39.6%calcium chloride, 50.4% sodium sulfate XIV 10.0% maltodextrin, 58.6%calcium lactate, 31.4% sodium chloride

-   -   Table 4 provides expected final dry powder compositions. These        compositions are based on the expectation that the ion exchange        reaction described above goes to completion for Formulations I        and III. Without wishing to be bound by any particular theory,        the evaporation of the droplet that occurs during spray drying        is expected to drive the least soluble salt to precipitate        first, which is the calcium citrate and calcium sulfate in        formulations I and III, respectively.

TABLE 4 Dry Powder Products of Spray Drying Formulation Composition(w/w) I 10.0% leucine, 52.8% calcium citrate, 37.2% sodium chloride II10.0% leucine, 58.6% calcium lactate, 31.4% sodium chloride III 10.0%leucine, 48.4% calcium sulfate, 41.6% sodium chloride XIV 10.0%maltodextrin, 58.6% calcium lactate, 31.4% sodium chloride

Description of Placebo:

A Placebo formulation comprising 100 weight percent leucine was producedby spray drying. An aqueous phase was prepared for a batch process bydissolving leucine in ultrapure water with constant agitation until thematerials were completely dissolved in the water at room temperature.For a static mixing process, the ultrapure water was divided in half andhalf of the total required leucine was dissolved in each volume ofwater. The solutions were then spray dried using a Niro or a Büchi spraydryer. For the Placebo formulation, two batches (A and B) of feedstockwere prepared and spray dried. The total solids concentration for BatchA was 15 g/L and for Batch B was 5 g/L. The process conditions used forspray drying Batch A (Placebo-A) on the Niro Mobile Minor spray dryerwere similar to the conditions used to spray dry Formulation I-A inExample 1. The process conditions used for spray drying Batch B(Placebo-B) were similar to the conditions used to spray dry FormulationI-C in Example 1, with the exception that the outlet temperature wasabout 82° C. for Formulation Placebo-B. Additional information relatingto process conditions and properties of the Formulation Placebo-A andPlacebo-B powders and/or particles prepared in this example are providedin the Tables or graphs shown in FIGS. 1A-1F and 2-4.

Example 1

This example describes the preparation of dry powders using feedstock ofFormulation I: 10.0 weight percent leucine, 35.1 weight percent calciumchloride and 54.9 weight percent sodium citrate.

An aqueous phase was prepared for a batch process by dissolving leucinein ultrapure water, then sodium citrate dihydrate, and finally calciumchloride dihydrate. The solution or suspension was kept agitatedthroughout the process until the materials were completely dissolved inthe water at room temperature. For a static mixing process, the sodiumsalt and calcium salt were kept in separate solutions. The ultrapurewater was divided in half and half of the total required leucine wasdissolved in each volume of water. The sodium citrate dihydrate wasdissolved in one aqueous phase and the calcium chloride dihydratedissolved in the second aqueous phase. The solutions or suspensions werekept agitated throughout the process until the materials were completelydissolved in the water at room temperature. The solutions or suspensionswere then spray dried using a Niro or a Büchi spray dryer. For eachformulation, three batches (A, B & C) of feedstock were prepared andspray dried. Details on the liquid feedstock preparations for each ofthe three batches are shown in Table 5, where the total solidsconcentration is reported as the total of the dissolved anhydrousmaterial weights. Batch A particles were prepared using batch Afeedstock on a Niro spray dryer. Batch B and C particles were preparedusing the corresponding feedstocks on a Büchi spray dryer.

TABLE 5 Summary of liquid feedstock preparations of four batches ofparticles for Formulation I. Formulation: I-A I-B I-C I-D Liquidfeedstock mixing Static Batch Batch Static mixed mixed mixed mixed Totalsolids 10 g/L 5 g/L 5 g/L 15 g/L concentration Total solids 380 g 6.25 g10.50 g 570 g Total volume water 38.0 L 1.25 L 2.1 L 38 L Amount leucinein 1 L 1.00 g 0.50 g 1.05 g 1.5 g Amount sodium citrate 6.26 g 3.13 g3.13 g 9.39 g dihydrate in 1 L Amount calcium chloride 4.65 g 2.32 g2.32 g 6.98 g dihydrate in 1 L

Batch A (I-A) dry powders were produced by spray drying on the NiroMobile Minor spray dryer (GEA Process Engineering Inc., Columbia, Md.)with powder collection from a product cartridge filter. Atomization ofthe liquid feed used a co-current two-fluid nozzle from Niro (GEAProcess Engineering Inc., Columbia, Md.) with 1.0 mm insert. The liquidfeed was fed using gear pumps (Cole-Panner Instrument Company, VernonHills, Ill.) into a static mixer (Charles Ross & Son Company, Hauppauge,N.Y.) immediately before introduction into the two-fluid nozzle.Nitrogen was used as the drying gas. The process gas inlet temperaturewas set to 282° C., with the outlet temperature reading about 98° C. Thegas supplying the two-fluid atomizer was set at a flowrate of 14.5 kg/hrand a pressure of 2 psi, the process gas flowrate was set at 85 kg/hrand a pressure of 25 psi, and the pressure inside the drying drum was at−2″ WC. The liquid feed stock total flowrate was 70 mL/min, with eachstream being fed at 35 mL/min Spray dried powders were collected from aproduct collection cartridge filter.

Batch B (I-B) and Batch C (I-C) dry powders were prepared by spraydrying on a Büchi B-290 Mini Spray Dryer (BÜCHI Labortechnik AG, Flawil,Switzerland) with a Büchi two-fluid nozzle with a 1.5 mm diameter andpowder collection from a High Performance cyclone. The system used theBüchi B-296 dehumidifier to ensure stable temperature and humidity ofthe air used to spray dry. Inlet temperature of the process gas was setat 220° C. with a liquid feedstock flowrate of 6.7 mL/min forFormulation I-B and 7 mL/min for Formulation I-C. The outlet temperaturewas about 108° C. for Formulation I-B and about 95° C. for FormulationI-C. The two-fluid atomizing gas was at 40 mm and the aspirator rate at90%.

Batch D (I-D) dry powders were produced by spray drying on the NiroMobile Minor spray dryer (GEA Process Engineering Inc., Columbia, Md.)with powder collection from a product filter membrane. Atomization ofthe liquid feed used a two-fluid nozzle from Spraying Systems (CarolStream, Ill.) with gas cap 67147 and fluid cap 2850SS. The liquid feedwas fed using gear pumps (Cole-Panner Instrument Company, Vernon Hills,Ill.) into a static mixer (Charles Ross & Son Company, Hauppauge, N.Y.)immediately before introduction into the two-fluid nozzle. Nitrogen wasused as the drying gas. The process gas inlet temperature was set toapproximately 265° C., with the outlet temperature reading about 99° C.The gas supplying the two-fluid atomizer was set at a flowrate of 80g/min, the process gas flowrate was set at 80 kg/hr and the pressureinside the drying drum was at −2″ WC. The liquid feed stock totalflowrate was 66 mL/min, with each stream being fed at 33 mL/min Spraydried powders were collected from a product collection filter membrane.

Some of the physical properties of the particles obtained in fourseparate batches (Formulation I-A, I-B, I-C and I-D) are summarized inTable 6. In addition to the data provided in Table 5, further datarelated to the dry powders prepared from feedstock formulation I-A issummarized as follows. The fine particle fraction (FPF) as measured by afull 8-stage Andersen Cascade Impactor with gravimetric analysis was onaverage 56.2% for FPF less than 5.6 microns and 41.7% for FPF less than3.4 microns. The aerodynamic diameter was also measured with afull-stage ACI with gravimetric analysis. The average value for the massmedian aerodynamic diameter (MMAD) was 2.72 microns. The volume size wasdetermined by laser diffraction on the HELOS/RODOS sizing equipment andthe average value for the volume median diameter (×50) at a pressure of1 bar was 2.57 microns. In addition, the powder displayed relativelyflowrate independent behavior as can be seen from the ratio of ×50measured at 0.5 bar to ×50 measured at 4.0 bar, which was 1.19. Thevalue for 1/4 bar for these particles was 1.17.

Additional properties of the dry powders prepared from feedstockFormulation I-D are summarized as follows. The fine particle fraction(FPF) as measured by a full 8-stage Andersen Cascade Impactor withgravimetric analysis was on average 58.8% for FPF less than 5.6 micronsand 46.7% for FPF less than 3.4 microns. The aerodynamic diameter wasalso measured with a full-stage ACI with gravimetric analysis. Theaverage value for the mass median aerodynamic diameter (MMAD) was 2.38microns. The volume size was determined by laser diffraction on theHELOS/RODOS sizing equipment and the average value for the volume mediandiameter (×50) at a pressure of 1 bar was 2.45 microns. In addition, thepowder displayed relatively flowrate independent behavior as can be seenfrom the ratio of ×50 measured at 0.5 bar to ×50 measured at 4.0 bar,which was 1.12. The value for 1/4 bar for these particles was 1.09.

TABLE 6 Summary of ACI-2 data for the four batches of particles forFormulation I. Formulation: I-A I-B I-C I-D FPF less than 5.6 μm onACI-2 (%) 61.6 49.2 64.8 67.2 FPF less than 3.4 μm on ACI-2 (%) 45.733.3 52.1 54.8

Additional information relating to properties of the Formulation I-Apowder and/or particles prepared in this example are provided in theTables or graphs shown in FIGS. 1A-1F and 2-4. In FIG. 1D, GSD refers togeometric standard deviation. In FIG. 1F, Dv50 refers to volume mediangeometric diameter (VMGD) as measured by Spraytec instrument; V refersto volume. SEM was performed as described above (FIG. 5A).

Example 2

This example describes the preparation of dry powders using feedstock ofFormulation II: 10.0 weight percent leucine, 58.6 weight percent calciumlactate and 31.4 weight percent sodium chloride.

An aqueous phase was prepared for a batch process by dissolving leucinein ultrapure water, then sodium chloride, and finally calcium lactatepentahydrate. The solution was kept agitated throughout the processuntil the materials were completely dissolved in the water at roomtemperature. For the calcium lactate formulation, four batches (A, B, Cand D) of feedstock were prepared and spray dried. Details on the liquidfeedstock preparations for each of the four batches are shown in Table7, where the total solids concentration is reported as the total of thedissolved anhydrous material weights. Batch A and D particles wereprepared using batch A and D feedstock, respectively on a Niro spraydryer. The process conditions used for spray drying Batch A (II-A) weresimilar to the conditions used to spray dry Formulation I-A in Example 1and those for Batch D (II-D) were similar to the conditions used tospray dry Formulation 1-D in Example 1. Batch B and C particles wereprepared using the corresponding feedstocks on a Büchi Mini spray dryerwith process conditions similar to those used to spray dry forFormulations I-B and I-C in Example 1, with the exception of thefollowing process conditions. The liquid feedstock flowrate was set at5.2 mL/min for Formulation II-B and 6 mL/min for Formulation II-C. Theoutlet temperature was about 91° C. to 109° C. for Formulation II-B andabout 100° C. for Formulation II-C.

TABLE 7 Summary of liquid feedstock preparations of four batches ofparticles for Formulation II. Formulation: II-A II-B II-C II-D Liquidfeedstock mixing Static Batch Batch Static mixed mixed mixed mixed Totalsolids 10 g/L 5 g/L 5 g/L 15 g/L concentration Total solids 400 g 10.0 g9.20 g 570 g Total volume water 40.0 L 2.00 L 1.84 L 38 L Amount leucinein 1 L 1.00 g 0.50 g 0.50 g 1.5 g Amount sodium 3.14 g 1.57 g 1.57 g4.71 g chloride in 1 L Amount calcium lactate 8.28 g 4.13 g 4.13 g 12.42g pentahydrate in 1 L

Some of the physical properties of the particles obtained in fourseparate batches (Formulation II-A, II-B, II-C and II-D) are summarizedin Table 8. In addition to the data provided in Table 8, further dataabout the dry particles prepared by feedstock formulation II-A issummarized as follows. The fine particle fraction (FPF) as measured by afull 8-stage Andersen Cascade Impactor with gravimetric analysis was onaverage 55.3% for FPF less than 5.6 microns and 39.7% for FPF less than3.4 microns. The aerodynamic diameter was also measured with afull-stage ACI with gravimetric analysis. The average value for the massmedian aerodynamic diameter (MMAD) was 2.89 microns. The volume size wasdetermined by laser diffraction on the HELOS/RODOS sizing equipment andthe average value for the volume median diameter (×50) at a pressure of1 bar was 1.51 microns. In addition, the powder displayed relativelyflowrate independent behavior as can be seen from the ratio of ×50measured at 0.5 bar to ×50 measured at 4.0 bar, which was 1.12. Thevalue for 1/4 bar for these particles was 1.08.

Additional properties of the dry powders prepared by feedstockformulation II-D are summarized as follows. The fine particle fraction(FPF) as measured by a full 8-stage Andersen Cascade Impactor withgravimetric analysis was on average 62.2% for FPF less than 5.6 micronsand 45.3% for FPF less than 3.4 microns. The aerodynamic diameter wasalso measured with a full-stage ACI with gravimetric analysis. Theaverage value for the mass median aerodynamic diameter (MMAD) was 2.72microns. The volume size was determined by laser diffraction on theHELOS/RODOS sizing equipment and the average value for the volume mediandiameter (×50) at a pressure of 1 bar was 1.47 microns. In addition, thepowder displayed relatively flowrate independent behavior as can be seenfrom the ratio of ×50 measured at 0.5 bar to ×50 measured at 4.0 bar,which was 1.08. The value for 1/4 bar for these particles was 1.03.

TABLE 8 Summary of ACI-2 data for the four batches of particles forFormulation II. Formulation: II-A II-B II-C II-D FPF less than 5.6 μm onACI-2 (%) 63.5 55.4 56.5 71.4 FPF less than 3.4 μm on ACI-2 (%) 43.435.5 34.7 49.7

Additional information relating to properties of the Formulation IIpowders and/or particles prepared in this example are provided in theTables or graphs shown in FIGS. 1A-1F and 2-4. SEM was performed asdescribed above (FIG. 5B).

Example 3

This example describes the preparation of dry powders using feedstock ofFormulation III: 10 weight percent leucine, 39.6 weight percent calciumchloride and 50.4 weight percent sodium sulfate.

An aqueous phase was prepared for a batch process by dissolving leucinein ultrapure water, then sodium sulfate, and finally calcium chloridedihydrate. The solution or suspension was kept agitated throughout theprocess until the materials were completely dissolved in the water atroom temperature. For a static mixing process, the sodium salt andcalcium salt were kept in separate solutions. The ultrapure water wasdivided in half and half of the total required leucine was dissolved ineach volume of water. The sodium sulfate was dissolved in one aqueousphase and the calcium chloride dihydrate dissolved in the second aqueousphase. The solutions or suspensions were kept agitated throughout theprocess until the materials were completely dissolved in the water atroom temperature. The solutions or suspensions were then spray driedusing a Niro or a Büchi spray dryer. For each formulation, four batches(A, B, C and D) of feedstock were prepared and spray dried. Details onthe liquid feedstock preparations for each of the four batches are shownin Table 9, where the total solids concentration is reported as thetotal of the dissolved anhydrous material weights. Batch A and Dparticles were prepared using batch A and D feedstock, respectively on aNiro spray dryer. Batch B and C particles were prepared using thecorresponding feedstocks on a Büchi spray dryer. The process conditionsused for spray drying Batch A (III-A) were similar to the conditionsused to spray dry Formulation I-A in Example 1 and the processconditions used for spray drying Batch D (III-D) were similar to theconditions used to spray dry Formulation I-D in Example 1. Batch B and Cparticles were prepared using the corresponding feedstocks on a BüchiMini spray dryer with process conditions similar to those used to spraydry Formulations I-B and I-C in Example 1, with the exception of thefollowing process conditions. The liquid feedstock flowrate was set at8.3 mL/min for Formulation M-B and 7 mL/min for Formulation III-C. Theoutlet temperature was about 83° C. for Formulation III-B and about 92°C. for Formulation III-C. The aspirator was set at 80% for FormulationIII-B.

TABLE 9 Summary of liquid feedstock preparations of four batches ofparticles for Formulation III. Formulation: III-A III-B III-C III-DLiquid feedstock mixing Static Batch Batch Static mixed mixed mixedmixed Total solids 10 g/L 5 g/L 5 g/L 15 g/L concentration Total solids400 g 2.5 g 9.5 g 185 g Total volume water 40 L 0.5 L 1.9 L 37 L Amountleucine in 1 L 1.00 g 0.5 g 0.5 g 0.5 g Amount sodium 5.04 g 2.52 g 2.52g 2.52 g sulfate in 1 L Amount calcium 5.25 g 2.61 g 2.61 g 2.61 gchloride dihydrate in 1 L

The physical properties of the particles obtained in four separatebatches (Formulation III-A, III-B, III-C and III-D) are summarized inTable 10. In addition to the data provided in Table 10, further dataabout the dry powders prepared from feedstock formulation III-A issummarized as follows. The fine particle fraction (FPF) as measured by afull 8-stage Andersen Cascade Impactor with gravimetric analysis was onaverage 68.7% for FPF less than 5.6 microns and 51.5% for FPF less than3.4 microns. The aerodynamic diameter was also measured with afull-stage ACI with gravimetric analysis. The average value for the massmedian aerodynamic diameter (MMAD) was 2.59 microns. The volume size wasdetermined by laser diffraction on the HELOS/RODOS sizing equipment andthe average value for the volume median diameter (×50) at a pressure of1 bar was 2.50 microns. In addition, the powder displayed relativelyflowrate independent behavior as can be seen from the ratio of ×50measured at 0.5 bar to ×50 measured at 4.0 bar, which was 1.47. Thevalue for 1/4 bar for these particles was 1.42.

Additional properties of the dry powders prepared by feedstockformulation III-D are summarized as follows. The fine particle fraction(FPF) as measured by a full 8-stage Andersen Cascade Impactor withgravimetric analysis was on average 77.9% for FPF less than 5.6 micronsand 68.3% for FPF less than 3.4 microns. The aerodynamic diameter wasalso measured with a full-stage ACI with gravimetric analysis. Theaverage value for the mass median aerodynamic diameter (MMAD) was 2.17microns. The volume size was determined by laser diffraction on theHELOS/RODOS sizing equipment and the average value for the volume mediandiameter (×50) at a pressure of 1 bar was 1.90 microns. In addition, thepowder displayed relatively flowrate independent behavior as can be seenfrom the ratio of ×50 measured at 0.5 bar to ×50 measured at 4.0 bar,which was 1.17. The value for 1/4 bar for these particles was 1.63.

TABLE 10 Summary of ACI_2 data for the four batches of particles forFormulation III. Formulation: III-A III-B III-C III-D FPF less than 5.6μm on ACI-2 (%) 82.7 62.0 69.0 82.8 FPF less than 3.4 μm on ACI-2 (%)60.1 47.4 53.2 70.9

Additional information relating to properties of the Formulation IIIpowders and/or particles prepared in this example is provided in theTables or graphs shown in FIGS. 1A-1F and 2-4. SEM was performed asdescribed above (FIG. 5C)

Example 4

This example describes the dose emission of powders of formulationbatches I-B, II-B, and III-B from dry powder inhaler at room andelevated conditions.

Method: Spray dried powders of the three different formulations (I-B,II-B, and III-B) were filled into size 2 HPMC capsules (Quali-V,Qualicaps, Whitsett, N.C.) to approximately half full (13-30 mgdepending on powder). Capsules were punctured prior to loading into oneof four capsule DPIs in order to ensure adequate hole openings in thecapsule. The capsules were loaded horizontally into the inhalers whichwere then connected to the custom chamber. Each dry powder inhaler had apressure transducer connected to it to monitor the flow rate through theinhaler during the test. When the test was begun, an airflow of 45 L/minwas drawn through each inhaler for 3 short bursts of 0.3 seconds each,separated by 1 minute. During each burst, the air drawn through theinhaler caused the capsule to spin and emit the powder in it into one of4 sub-chambers which had one row of 3 tissue culture wells forming thefloor of the sub-chamber. The aerosol cloud was allowed to settle forone minute before the next subsequent burst for a total of 3 bursts anda total air volume of 0.68 L being drawn through the inhaler. Theduration and total airflow rate was controlled with a flow controller(TPK-2000, MSP Corporation, Shoreview, Minn.) and recorded with an airmass flow meter (model#3063, TSI Inc., Shoreview, Minn.). Individualinhaler airflow rates were monitored with pressure sensors (model#ASCX01DN, Honeywell International Inc., Morristown, N.J.) which hadbeen previously calibrated and whose signal was converted to flow ratevia a custom Lab-view code. In one case, the custom chamber was locatedon the lab bench at room conditions, while in another 2 cases it waslocated in a stability chamber (Darwin Chambers Company, St. Louis, Mo.)set to 37° C. and 90% RH. For the first case in the stability chamber,the capsules were punctured and loaded into inhalers at room conditions,the door of the chamber was opened, the inhalers attached and the flowrate was actuated ˜30 seconds after the capsules entered the chamber. Inthe second case, the capsules were first placed unpunctured in thestability chamber for 3 minutes, then removed from the chamber,punctured and loaded at room conditions, attached in the chamber andactuated within 30 seconds of the second entry into the chamber.Following each test, the capsules were removed from the inhalers andweighed and used to calculate the percentage of powder emitted from thecapsule. For each of the 3 sets of conditions, two 12 well tissueculture plates (each plate required 4 capsules in 4 inhalers deliveringpowder to 3 wells each) were exposed to powder for each of the powderformulations tested, giving a total of 8 capsule emissions for eachpowder at each temperature and humidity setting.

As shown in Table 11 below, for all three powder batches (I-B, B-B, andIII-B) the average amount of powder emitted from the capsule is greaterthan 99% based on the weight change of the capsule.

TABLE 11 Emitted Dose Percent Powder Batch Emitted Dose % I-B 99.45 II-B100.0 III-B 99.38

Example 5

This example describes the dispersion properties and density propertiesof formulations I-A, II-A, III-A, and Leucine formulation for placebo assummarized in Table 12. All the data found in Table 12 can also be foundin FIGS. 1A through 1E. As evidenced by the results shown in Table 12,all formulations are highly dispersible, meaning that their measuredvolume sizes are relatively independent of pressure on the HELOS/RODOS.As shown in Table 12, the ratio of the volume median sizes obtained atlow dispersion pressures (0.5 bar or 1.0 bar) and at a high dispersionpressure (4.0 bar) can be used as an indicator of dispersibility. Thesevalues are referred to as the 0.5 bar/4.0 bar ratio or the 1.0 bar/4.0bar ratio.

The tap density was determined by the modified USP<616> method using a1.5 cc microcentrifuge tube and the average value for tap density at1,000 taps were 0.29, 0.69, 0.34, and 0.04 g/cc, respectively. The MMAD,as measured by a full-stage (eight-stage) Andersen Cascade Impactor(ACI), were 2.72, 2.89, 2.59, and 4.29 μm, respectively. The FPF below3.4 μm, as measured on a full-stage ACI, were 41.7%, 39.7%, 51.5%, and17.4%, respectively, and below 5.6 μm were 56.2%, 55.3%, 68.7%, and32.5%, respectively. The volume size was determined by laser diffractionand the average values for the volume median diameter (×50) at apressure of 1 bar were 2.57 microns, 1.51 microns, 2.50 microns, and6.47 microns, respectively. Values for pressure values at 0.5 bar, 2.0bar, and 4.0 bar can be seen in Table 12. In addition, the powderdisplayed relatively flowrate independent behavior as can be seen fromthe ratio of ×50 measured at 0.5 bar to ×50 measured at 4.0 bar as shownin Table 12. The values are 1.19, 1.12, 1.47, and 1.62, respectively.The table also includes values for the ratio of 1.0 bar to 4.0 bar, forthe sake of comparison to other art, since this is another measure offlowrate dependency.

TABLE 12 Dispersion and Density Properties of Formulations I-A, II-A,III-A Density Tap ACI-8, Gravimetric Spraytec HELOS/RODOS density MMADFPF_TD FPF_TD Dv50 Regulator x50 (g/cc) (um) <3.4 um <5.6 um (um)pressure (μm) 0.5 bar/ 1 bar/ Formulation Ave Ave Ave Ave Ave (bar) Ave4 bar 4 bar Formulation 0.29 2.72 41.7% 56.2% 3.07 0.5 2.62 1.19 1.17I-A 1.0 2.57 2.0 2.49 4.0 2.20 Formulation 0.69 2.89 39.7% 55.3% 1.780.5 1.57 1.12 1.08 II-A 1.0 1.51 2.0 1.47 4.0 1.40 Formulation 0.34 2.5951.5% 68.7% 3.05 0.5 2.59 1.47 1.42 III-A 1.0 2.50 2.0 2.17 4.0 1.76Placebo 0.04 4.29 17.4% 32.5% 21.77 0.5 7.68 1.62 1.37 (100% 1.0 6.47leucine) 2.0 5.69 4.0 4.74

Example 6

This example describes the preparation of dry powders using feedstockFormulations 6.1-6.9 as listed in Table 13 below.

TABLE 13 Feedstock Formulations 6.1-6.9 Formulation Composition andWeight % (w/w) 6.1 10.0% leucine, 58.6% calcium lactate, 31.4% sodiumchloride 6.2 50.0% leucine, 48.4% calcium lactate, 1.6% sodium chloride6.3 10.0% leucine, 66.6% calcium lactate, 23.4% sodium chloride 6.410.0% leucine, 35.1% calcium chloride, 54.9% sodium citrate 6.5 67.1%leucine, 30.0% calcium chloride, 2.9% sodium citrate 6.6 39.0% calciumchloride, 61.0% sodium citrate 6.7 10.0% leucine, 39.6% calciumchloride, 50.4% sodium sulfate 6.8 67.6% leucine, 30.0% calciumchloride, 2.4% sodium sulfate 6.9 44.0% calcium chloride, 56.0% sodiumsulfate

The general mode of preparation of the dry powders in this example issimilar to what was described for the powders in the above examples withthe exception that all of the dry powders in this example were spraydried using a Büchi B-290 spray dryer with High Performance cyclone.Formulations 6.1, 6.4, and 6.7 in this Example correspond toFormulations II-B, I-B, and III-B in the Examples above, respectively.

The physical properties of the powders and/or particles obtained in thisexample are summarized in the Tables shown in FIGS. 6A and 6B.Formulations 6.1-6.9 in Table 13 correspond to Formulations 6.1-6.9 inFIGS. 6A and 6B, respectively. In FIG. 6A, ×50 and Dv50 refer to volumemedian diameter or volume median geometric diameter (VMGD); and GSDrefers to geometric standard deviation. In FIG. 6B, yield % refers topercentage of the weight of the recovered product in the collection jarattached to the High Performance cyclone divided by the weight of thesolutes in the feedstock. All other abbreviations are describedelsewhere in the application.

Example 7

This example describes the dose emission of powders prepared byfeedstock Formulations 6.1-6.9 from a dry powder inhaler at room andelevated conditions. Some of this data is also presented above, inExample 4.

Method: Spray dried powders of the nine feedstock formulations 6.1-6.9were separately filled into size 2 HPMC capsules (Quali-V, Qualicaps,Whitsett, N.C.) to approximately half full (13-30 mg depending onpowder). Capsules were punctured prior to loading into one of fourcapsule based DPIs in order to ensure adequate hole openings in thecapsule. The capsules were loaded horizontally into the inhalers whichwere then connected to the custom chamber. Each dry powder inhaler had apressure transducer connected to it to monitor the flow rate through theinhaler during the test. When the test was begun, an airflow of 45 L/minwas drawn through each inhaler for 3 short bursts of 0.3 seconds each,separated by 1 minute. During each burst, the air drawn through theinhaler caused the capsule to spin and emit the powder in it into one of4 sub-chambers which had one row of 3 tissue culture wells forming thefloor of the sub-chamber. The aerosol cloud was allowed to settle forone minute before the next subsequent burst for a total of 3 bursts anda total air volume of 0.68 L being drawn through the inhaler. Theduration and total airflow rate was controlled with a flow controller(TPK-2000, MSP Corporation, Shoreview, Minn.) and recorded with an airmass flow meter (model#3063, TSI Inc., Shoreview, Minn.). Individualinhaler airflow rates were monitored with pressure sensors (model#ASCX01DN, Honeywell International Inc., Morristown, N.J.) which hadbeen previously calibrated and whose signal was converted to flow ratevia a custom Lab-view code. In one case, the custom chamber was locatedon the lab bench at room conditions, while in another 2 cases it waslocated in a stability chamber (Darwin Chambers Company, St. Louis, Mo.)set to 37° C. and 90% RH. For the first case in the stability chamber,the capsules were punctured and loaded into inhalers at room conditions,the door of the chamber was opened, the inhalers attached and the flowrate was actuated ˜30 seconds after the capsules entered the chamber. Inthe second case, the capsules were first placed unpunctured in thestability chamber for 3 minutes, then removed from the chamber,punctured and loaded at room conditions, attached in the chamber andactuated within 30 seconds of the second entry into the chamber.Following each test, the capsules were removed from the inhalers andweighed and used to calculate the percentage of powder emitted from thecapsule. For each of the 3 sets of conditions, two 12 well tissueculture plates (each plate required 4 capsules in 4 inhalers deliveringpowder to 3 wells each) were exposed to powder for each of the powderformulations tested, giving a total of 8 capsule emissions for eachpowder at each temperature and humidity setting.

As shown in Table 14 below, for all nine powder batches (obtained usingfeedstock Formulations 6.1-6.9) the average amount of powder emittedfrom the capsule is greater than 98% based on the weight change of thecapsule.

TABLE 14 Emitted Dose Percent Formulation Emitted Dose (%) 6.1 100.00%6.2 98.86% 6.3 99.85% 6.4 99.45% 6.5 99.68% 6.6 100.00% 6.7 99.38% 6.898.05% 6.9 100.00%

Example 8

This example describes the results of a short-term stability study thatwas conducted for the dry powders prepared by feedstock formulations6.1, 6.4 and 6.7.

An important characteristic of pharmaceutical dry powders is stabilityat different temperature and humidity conditions. One property that maylead to an unstable powder is the powder's tendency to absorb moisturefrom the environment, which then will likely lead to agglomeration ofthe particles, thus altering the apparent particle size of the powder atsimilar dispersion conditions. Spray dried powders were held at a rangeof conditions for a periods of one week to three or more months andperiodically tested for particle size distribution. Storage conditionsincluded closed capsules in vials at 25° C. and 60% RH, closed capsulesin vials at 40° C. and 75% RH, closed capsules at room temperature and40% RH, open capsules at 30° C. and 65% RH and open capsules at 30° C.and 75% RH. Size 3 HPMC capsules (Quali-V, Qualicaps, Whitsett, N.C.)were half filled with each dry powder. One sample was tested immediatelyin the Spraytec (Malvern Instruments Inc., Westborough, Mass.), a laserdiffraction spray particle sizing system where dry powders can bedispersed from an inhaler using the inhaler cell setup. Approximately 16capsules were filled with each powder prepared using feedstock solutions6.1, 6.4 and 6.7. Capsules were kept in the lab at controlled humidityand temperature conditions (˜23-28% RH), and also in the outside lab atvarying temperature and relative humidity (˜40-75% RH). Capsules kept atstorage conditions of 25° C. and 60% RH, 40° C. and 75% RH, 30° C. and65% RH and 30° C. and 75% RH were held in stability chambers (DarwinChambers Company, St. Louis, Mo.) set at those conditions. At specifictime points (ranging from 30 min to 3 months), one to three capsulesfrom each condition were tested on the Spraytec for geometric particlesize distribution and the ACI-2 for aerodynamic particle sizeproperties.

Generally, the powders that were in closed capsules in vials remainedstable for a long period of time, longer than three months. Powders thatwere in open capsules with no vials showed agglomeration after exposureto higher humidity conditions. The stability data are summarized inTable 15 below.

TABLE 15 Short-term Stability Data closed capsules closed capsules, opencapsules, no vials in vials no vials Spraytec ACI-2 25 C./ SpraytecACI-2 30 C./65% ACI-2 Spraytec 30 C./75% Formulation CounterionExcipient 60% RH 40 C./75% RH 40% RH 40% RH RH 30 C./65% RH 30 C./75% RHRH 6.1 Lactate 10% >3 months 0.5-1 month >8 days 4-6 days >30 min >30min >30 min >30 min Leucine 6.4 Citrate 10% >3 months   1-3 months >7days N/A >30 min >30 min <30 min >30 min Leucine 6.7 Sulfate 10% >3months   1-3 months 2-7 days  N/A >30 min >30 min >30 min >30 minLeucine

Example 9

This example describes a Bacterial Pass-Through Assay performed usingdry powders prepared using feedstock formulations A-E.

Method: To test the effect of aerosolized dry powder formulations onbacterial movement across mucus, a pass-through model was used. In thismodel, 200 μL of 4% sodium alginate (Sigma-Aldrich, St. Louis, Mo.) wasadded to the apical surface of a 12 mm Costar Transwell membrane(Corning, Lowell, Mass.; 3.0 μm pore size) and subsequently exposed todry powder formulations. Dry powders were aerosolized into the chamberusing a dry powder insufflator (Penn-Century, Inc., Philadelphia, Pa.)and allowed to settle by gravity over a 5 minute period. Following thisexposure, 10 μL of Klebsiella pneumoniae (˜10⁷ CFU/mL in saline) wasadded to the apical surface of the mimetic. At various time points afterthe addition of bacteria, aliquots of the basolateral buffer wereremoved and the number of bacteria in each aliquot was determined byserially diluting and plating on blood agar plates. A schematic of thismethod is shown in FIG. 7. The concentration of salt that was deliveredto each Transwell was quantified by HPLC. For this purpose, empty wellsof the 12 well cell culture plate that were next to each Transwell andwere exposed to the same dose of formulation were rinsed with sterilewater and diluted 1:1 with acetic acid to solubilize the calcium saltsin each powder.

The effect of calcium containing powders on K. pneumoniae movementthrough sodium alginate mucus mimetic was tested. Dry powderformulations comprising calcium salts with different solubilityprofiles, together with leucine and sodium chloride, were screened foractivity. Table 16 (below) lists the feedstock formulations of thepowders that were tested. A 50.0% (w/w) leucine loading in thecomposition was necessary, as opposed to the 10.0% (w/w) leucine loadingin the formulations described in the examples above, due to dosing anddetection limitations in the pass through model. The calcium and sodiummolar ratio was chosen for each formulation to target a 1:1 molar ratio,while not needing to go too low on the relative weights of anyparticular salt. Therefore, the lactate, citrate, and acetateformulations used were not in a 1:1 molar ratio in order to keep theweights of the sodium chloride and the calcium chloride in thoseformulations, respectively, above about 10% by weight.

TABLE 16 Feedstock Formulations Ca:Na mole Formulation Composition (w/w)ratio A 50.0% leucine, 1.0:2.0 22.0% calcium chloride, 28.0% sodiumsulfate B 50.0% leucine, 1.0:2.0 25.5% calcium chloride, 24.5% sodiumcarbonate C 50.0% leucine, 1.0:2.0 19.5% calcium chloride, 30.5% sodiumcitrate D 50.0% leucine, 1.0:1.3 37.0% calcium lactate, 13.0% sodiumchloride E 50.0% leucine, 1.0:1.8 33.75% calcium acetate, 16.25% sodiumchloride

The results for this test are shown in FIGS. 8A and 8B. The twodifferent figures represent two different sets of experiments, run atthe same conditions. The leucine control and sulfate data allow forrelative comparison between the two sets of experiments. The powderscontaining the anions sulfate, lactate, and acetate, i.e., the drypowders prepared from feedstock formulations A, D, and E, respectively,reduced the movement of bacteria across the mimetic, whereas the powderscontaining the anions carbonate and citrate, i.e., dry powders preparedfrom feedstock formulations B and C, exhibited no effect. These findingcorrelated with the known solubility of the calcium salts in water,suggesting that the possible failure of carbonate and citrate salts toinhibit the movement of K. pneumoniae could be related to the solubilityof these powders at the surface of the sodium alginate mimetic. Thisconclusion is also based on the plausible assumption that the ionexchange reaction described previously goes to completion during spraydrying, and that the form of the calcium salt in Formulations A throughE is calcium sulfate, calcium carbonate, calcium citrate, calciumlactate, and calcium acetate, respectively. The solubility of thesesalts from least soluble to most soluble: calcium carbonate<calciumcitrate<calcium sulfate<calcium lactate<calcium acetate. (See Table 1above.)

Example 10

This example describes the performance of dry powders in reducing viralreplication utilizing a viral replication model.

In this example, a series of dose response studies with different drypowder prepared from feedstock formulations consisting of differentcalcium salts are described. Dry powders were made with leucine, acalcium salt (lactate or chloride), and sodium salt (chloride, sulfate,citrate or carbonate). Feedstock formulations listed 10-1, 10-2 and 10-3were spray dried on a Büchi B-290 mini spray dryer. The system used theBüchi B-296 dehumidifier to ensure stable temperature and humidity ofthe air used to spray dry. Feedstock Formulation 10-4 was spray dried ona Niro Mobile Minor Spray Dryer in an open cycle with nitrogen.

Four liquid feedstocks were prepared with the following components andratios (weight percentage) as listed in Table 17.

TABLE 17 Feedstock Formulations Feedstock Composition Ca:Na moleFormulation (w/w) Lot Number ratio 10-1 50.0% leucine, 45.6.1 1.0:1.337.0% calcium lactate, 13.0% sodium chloride 10-2 50.0% leucine,27.155.1 1.0:2.0 22.0% calcium chloride, 28.0% sodium sulfate 10-3 50.0%leucine, 27.156.1 1.0:2.0 19.5% calcium chloride, 30.5% sodium citrate10-4 50.0% leucine, 26.019.1 1.0:2.0 25.5% calcium chloride, 24.5%sodium carbonate

A 50.0% (w/w) leucine loading in the composition was necessary, asopposed to the 10.0% (w/w) leucine loading in the formulations describedin the examples above, due to dosing and detection limitations in theviral replication model. The calcium and sodium mole ratio was chosenfor each formulation to target a 1:1 molar ratio, while not needing togo too low on the relative weights of any particular salt. Therefore,the lactate and citrate formulations used were not in a 1:1 mole ratioin order to keep the weights of the sodium chloride and the calciumchloride in those formulations, respectively, above about 10% by weight.

Formulations 10-1, 10-2 and 10-3 were spray dried with feedstock solidsconcentrations of 5 g/L, while the exact amount of salts and excipientdissolved in ultrapure water and its specific volume varied. Thefollowing process settings were used: inlet temperature of 220° C.,liquid flow rate of approximately 10 mL/min, room conditions at23.2-24.6° C. and 19-21% RH, and dehumidifier air at 3-5° C. and 30% RH.The outlet temperature, cyclone and aspirator rate varied. Formulation10-1 was spray dried using a high performance cyclone with the aspiratorat 80% and an outlet temperature of 93° C. Dry powder formulations 10-2and 10-3 were made with the regular cyclone, an aspirator at 100% and anoutlet temperature of 111-115° C. Formulation 10-4 was spray dried witha solids concentration of 2.7 g/L and the following process settings:inlet temperature of 140° C., outlet temperature of 75° C., liquidfeedstock flowrate of 30 mL/min, process gas flowrate of 100 kg/hr,atomizer gas flowrate of 20 g/min and a spray drying drum chamberpressure of −2″ WC.

A cell culture model of Influenza infection was used to study theeffects of Formulations 1 through 4. Calu-3 cells (American Type CultureCollection, Manassas, Va.) were cultured on permeable membranes (12 mmTranswells; 0.4 μm pore size, Corning Lowell, Mass.) until confluent(the membrane was fully covered with cells) and air-liquid interface(ALI) cultures were established by removing the apical media andculturing at 37° C./5% CO₂. Cells were cultured for >2 weeks at ALIbefore each experiment. Prior to each experiment the apical surface ofeach Transwell was washed 3× with PBS (Hyclone, Logan, Utah). Calu-3cells were exposed to dry powders using a proprietary dry powdersedimentation chamber. In order to expose cells to equivalent doses ofcalcium, capsules were filled with different amounts of each powder. Thehigh, medium, and low fill weights were calculated based on matching theamount of calcium delivered by each powder (4.23 mg, 1.06 mg, and 0.35mg). For each dry powder condition tested, two capsules were weighed asempty, filled, and after exposure in order to determine emitted dose ofthe powder. Table 18 (below) shows the capsule fill weights before andafter exposure and the concentration of calcium delivered to cells asdetermined by HPLC measurements. Immediately after exposure, thebasolateral media (media on the bottom side of the Transwell) wasreplaced with fresh media. Triplicate wells were exposed to dry powdersfrom each feedstock formulation in each test. A second cell cultureplate was exposed to the same dry powders from the feedstockformulations to quantify the delivery of total salt or calcium to cells.One hour after exposure, cells were infected with 10 μL of InfluenzaA/WSN/33/1 (H1N1) or Influenza A/Panama/2007/99 (H3N2) at a multiplicityof infection of 0.1-0.01 (0.1-0.01 virions per cell). Four hours afteraerosol treatment, the apical surfaces were washed to remove excess drypowders and unattached virus and cells were cultured for an additional20 h at 37° C. plus 5% CO₂. Twenty-four hours after aerosol treatment,virus released onto the apical surface of infected cells was collectedin culture media or PBS and the concentration of virus in the apicalwash was quantified by TCID₅₀ (50% Tissue Culture Infectious Dose)assay. The TCID₅₀ assay is a standard endpoint dilution assay that isused to quantify how much of a virus is present in a sample.

TABLE 18 Dry powder, prepared from feedstock formulations 10-1 to 10-4,tested to evaluate their effect on Influenza A/WSN/33/1 infection in acell culture model. Dry powder formulations were tested to evaluatetheir effect on Influenza A/WSN/33/1 infection in a cell culture model.To deliver an equivalent amount of calcium ion (Ca⁺²), the desired fillweight was calculated for each dry powder formulation. Qualicap capsuleswere weighed empty, filled, and after exposure to determine the emitteddose. Triplicate wells were exposed to each capsule and after wells werewashed. HPLC analysis of these samples determined the amount of Ca⁺²delivered to cells. Feedstock Calcium ion Formula- Capsule concentrationtion Intended Empty Filled after determined (for Dry Fill CapsuleCapsule Exposure by HPLC Powders) (mg) (mg) (mg) (mg) (μg/cm²) 10-253.18 31.7 83.0 31.9 20.5 ± 0.7^(a) (50.0% 13.29 32.5 45.9 33.9 5.8^(b)leucine, 4.43 33.3 38.4 33.9 2.8^(b) 22.0% calcium chloride, 28.0%sodium sulfate) 10-1 62.17 64.972, 99.649, 64.994, 50.9 ± 1.1^(a) (50.0%63.122* 98.881* 63.679* leucine, 15.54 63.525 81.926 68.141 12.7 ±1.7^(a) 37.0% 5.18 62.453 67.796 62.49 4.0^(b) calcium lactate, 13.0%sodium chloride) 10-3 60.0 64.4 123.6 81.994 20.5 ± 5.7^(a) (50.0% 14.9964.0 78.5 65.388  7.6 ± 0.9^(a) leucine, 5.00 63.5 70.3 63.829  3.6 ±1.5^(a) 19.5% calcium chloride, 30.5% sodium citrate) 10-4 45.88 64.6104.7 66.685 28.1 ± 7.3^(a) (50.0% 11.47 61.5 72.0 63.186  8.1 ± 2.6^(a)leucine, 3.82 61.8 62.6 63.341 5.62 ± 2.7^(a) 25.5% calcium chloride,24.5% sodium carbonate) *denotes the use of two capsules in order toachieve desired fill weight. ^(a)denotes n = 3, ^(b)denotes n = 1

Example 10A

Dry powders, prepared from feedstock formulations 10-1 to 10-4, reduceInfluenza A/WSN/33/1 (H1N1) infection in a dose-dependent manner.

To test the effect of dry powder formulations on Influenza infection ina cell culture model Calu-3 cells were exposed to four different drypowder formulations each consisting of 50% leucine, a calcium salt andsodium chloride. Viral infection was assessed by quantifying the amountof viral replication over a 24 h period. The specific powders tested arelisted in Table 18 (above), and included carbonate, lactate, sulfate andcitrate salts. In an attempt to expose cells to equivalent amounts ofcalcium of each of the four calcium containing powders, capsules werefilled to appropriate fill weights prior to dosing. Cells exposed to noformulation (Air) were used as control cells.

As seen in FIG. 9, each powder exhibited a dose-responsive reduction ininfluenza infection; however, the magnitude of the effect was differentamong the four powders tested. At low calcium concentrations calciumlactate was most efficacious suggesting that it was the most potent ofthe powders tested. At higher concentrations of calcium, the calciumlactate and calcium citrate powders exhibited similar efficacy.Additional testing of the calcium citrate powder at even higherconcentrations may demonstrate that it is the most efficacious powder.The calcium sulfate powder exhibited an intermediate effect and wascomparable to calcium citrate at several concentrations. Calciumcarbonate had only a minimal effect on viral replication even at thehighest concentration (less than 10-fold). Of note, calcium carbonate isthe least soluble of the powders tested.

As shown in FIG. 9, the dry powders prepared for this reduce Influenzainfection in a dose-dependent manner. Calu-3 cells exposed to noformulation were used as a control and compared to Calu-3 cells exposedto dry powder formulations at different fill weights. The concentrationof virus released by cells exposed to each aerosol formulation wasquantified. Bars represent the mean and standard deviation of triplicatewells for each condition. Data were analyzed statistically by one wayANOVA and Tukey's multiple comparison post-test.

Example 10B

Dry powder, prepared from feedstock formulations 10-1 to 10-4 in Table19, reduce Influenza A/Panama/2007/99 (H3N2) infection in adose-dependent manner.

To extend these studies, the same powders were tested with a secondinfluenza strain [Influenza A/Panama/2007/99 (H3N2)]. Similar to Example10A, Calu-3 cells were exposed to four different dry powder formulationseach consisting of 50% leucine, a calcium salt and sodium chloride.Viral infection was assessed by quantifying the amount of viralreplication over a 24 h period. The specific powders tested are listedin Table 19 (below) and included carbonate, lactate, sulfate and citratesalts. In an attempt to expose cells to equivalent amounts of calcium ofeach of the four calcium containing powders, capsules were filled toappropriate fill weights prior to dosing. Cells exposed to noformulation (Air) were used as control cells.

As seen in FIG. 10, using this strain, similar efficacy was observed foreach powder: calcium lactate was the most efficacious, calcium citrateand calcium sulfate exhibited intermediate efficacy and the calciumcarbonate powder was only minimally efficacious. These data support thebroad activity of Ca:Na dry powders against multiple influenza strains.

TABLE 19 Dry powders, prepared from feedstock formulations 10-1 to 10-4,tested to evaluate their effect on Influenza A/Panama/99/2007 (H3N2)infec- tion in a cell culture model. To deliver equivalent amount ofCa⁺², the desired fill weight was calculated for each dry powderformulation. Qualicap capsules were weighed empty, filled, and afterexposure to determine the emitted dose. Triplicate wells were exposed toeach capsule and after wells were washed. HPLC analysis of these samplesdetermined the amount of Ca⁺² delivered to cells. Feedstock Calcium ionFormula- Capsule concentration tion Desired Empty Filled afterdetermined (for Dry Fill Capsule Capsule Exposure by HPLC Powders) (mg)(mg) (mg) (mg) (μg/cm² ± SD)^(a) 10-2 53.18 61.358 121.417 62.591 40.8 ±5.0  (50.0% 13.29 60.602 76.804 62.167 10.5 ± 2.3  leucine, 4.43 65.10270.789 65.670 2.9 ± 0.6 22.0% calcium chloride, 28.0% sodium sulfate)10-1 62.17 64.037 125.465 67.043 33.8 ± 3.5  (50.0% 15.54 65.358 82.47465.632 9.7 ± 1.4 leucine, 5.18 66.046 72.455 66.324 3.4 ± 0.9 37.0%calcium lactate, 13.0% sodium chloride) 10-3 60.0 62.581 108.035 63.84129.6 ± 10.1 (50.0% 14.99 63.393 75.770 64.085 8.1 ± 1.4 leucine, 5.0065.910 70.062 66.204 4.1 ± 0.8 19.5% calcium chloride, 30.5% sodiumcitrate) 10-4 45.88 64.506 115.876 65.004 30.4 ± 11.9 (50.0% 11.4764.319 77.627 65.080 11.1 ± 4.3  leucine, 3.82 66.495 71.398 66.698 2.4± 1.0 25.5% calcium chloride, 24.5% sodium carbonate)

As shown in FIG. 10, the dry powders prepared for this Example reduceInfluenza A/Panama/99/2007 (H3N2) infection in a dose-dependent manner.Calu-3 cells exposed to no formulation (0 μg Ca²⁺/cm²) were used as acontrol and compared to Calu-3 cells exposed to dry powder formulationsat different fill weights and therefore different concentrations ofcalcium. The concentration of calcium delivered to cells in eachexperiment for each fill weight was determined using HPLC measurementsof calcium in washes from empty plates exposed to each condition. Theconcentration of virus released by cells exposed to each aerosolformulation 24 h after dosing was quantified by TCID₅₀ assay. Each datapoint represents the mean and standard deviation of triplicate wells foreach condition.

Example 11 In Vivo Influenza Model

This example demonstrates that dry powder formulations comprised ofcalcium salts and sodium chloride reduce the severity of influenzainfection in ferrets. The formulations tested are shown in Table 20.Control ferrets were exposed to a powder comprised of 100% leucine underthe same exposure conditions. In preliminary in vitro studies, thiscontrol powder had no effect on viral replication. Calcium powders andcontrol (Formulation I lot: 26-190-F, Formulation III lot: 65-009-F,Formulation II lot: 65-003-F and Leucine lot: 65-017-F) were aerosolizedwith a Palas Rotating Brush Generator 1000 solid particle disperser(RBG, Palas GmbH, Karlsruhe, Germany). Ferrets (n=8 per group) wereexposed to ˜0.2 mg Ca/kg and the severity of infection was evaluatedover time. Each formulation was dispersed in a nose-only exposure system1 hour before infection, 4 hours after infection and then BID for 4 days(d1-4). The study was terminated on day 10. Body temperatures weredetermined twice a day beginning on day 0 of the study. Ferrets infectedwith influenza typically show increases in body temperature within 2days of infection, drop body weight over the course of the study andshow clinical signs of infection such as lethargy and sneezing. Thesechanges coincide with an increase in influenza viral titers shed fromthe nasal cavity and increases in nasal inflammation.

TABLE 20 Formulations tested for efficacy in ferrets FormulationComposition Formulation I 10.0% leucine, 35.1% calcium chloride, 54.9%sodium citrate (Active with 12.7% calcium ion) Formulation II 10.0%leucine, 39.6% calcium chloride, 50.4% sodium sulfate (Active with 14.3%calcium ion) Formulation III 10.0% leucine, 58.6% calcium lactate, 31.4%sodium chloride (Active with 10.8% calcium ion)

On study day −4, ferrets were implanted with a microchip subcutaneouslyin the right rear flank and another in the shoulder for redundancy. Thetransponder chip (IPTT-300 Implantable Programmable Temperature andIdentification Transponder; Bio Medic Data Systems, Inc, Seaford, Del.19973) allows for ferret identification and provides subcutaneous bodytemperature data throughout the study using a BMDS electronic proximityreader wand (WRS-6007; Biomedic Data Systems Inc, Seaford, Del.).Subcutaneous body temperatures taken on day −3 to −1 were used asbaseline temperatures and used to calculate the change from baseline foreach animal over the course of the study. Treatment with a dry powderformulation comprised of leucine (excipient), Ca-lactate (FormulationIII), and NaCl had a significant impact on body temperature increases(FIG. 11C). The mean body temperature changes in this group remained ator below baseline measurements for the course of the study and the areaunder the curve (AUC) measurements were approximately 5-fold lower thanthe control (FIG. 11D). The two other powders tested exhibited lesspronounced efficacy that was limited to differences from the control onspecific days of the study. In particular, both the Ca citrate and Casulfate treated groups had lower body temperatures than the controlanimals on day 3 of the study (FIGS. 11A and 11B, respectively) and theCa sulfate group had lower body temperatures over the final three daysof the study.

Example 12

This example demonstrates that dry powder formulations comprised ofdifferent excipients reduce influenza infection, but at higher dosesthan formulations comprised of leucine.

To assess the impact of the excipient on efficacy in vitro we tested twodry powder formulations (Table 21) that varied in excipient and comparedtheir efficacy to Formulation III (containing leucine) using theinfluenza replication model. These formulations contained the sameconcentration of calcium lactate and sodium chloride and the same weightpercentage of excipient (10%).

TABLE 21 Formulations used to evaluate efficacy against multipleinfluenza viruses and to test different excipients Ca:Na molar Manufac-Lot # Formulation Composition ratio turing 26-190-F Formulation I 10.0%leucine, 1:2 Niro 35.1% calcium chloride, 54.9% sodium citrate (Activewith 12.7% calcium ion) 65-003-F Formulation II 10.0% leucine, 1:2 Niro39.6% calcium chloride, 50.4% sodium sulfate (Active with 14.3% calciumion) 65-009-F Formulation III 10.0% leucine, 1:2 Niro 58.6% calciumlactate, 31.4% sodium chloride (Active with 10.8% calcium ion) 45.137.2N/A 10.0% mannitol, 1:2 Büchi 58.6% calcium lactate, 31.4% sodiumchloride (Active with 10.8% calcium ion) 45.137.3 Formulation XIV 10.0%maltodextrin, 1:2 Büchi 58.6% calcium lactate, 31.4% sodium chloride(Active with 10.8% calcium ion)

Calu-3 cells exposed to no formulation were used as a control andcompared to Calu-3 cells exposed to dry powder comprised of calciumlactate and sodium chloride with different excipients. Three differentfill weights of the mannitol and maltodextrin powders were used to covera dose range between 10 to 30 μg Ca2+/cm2. The concentration of virusreleased by cells exposed to each aerosol formulation was quantified(FIG. 12). Each data point represents the mean and standard deviation ofduplicate wells for each concentration. Data were analyzed by one-wayANOVA and Tukey's multiple comparisons post-test. The data for the lowdose of each powder is representative of two independent experiments.

Both the mannitol and maltodextrin containing formulations reducedinfluenza infection in a dose responsive manner, however, they weresignificantly less potent than the leucine containing powder. At a doseof 14.8 μg Ca^(2+/)cm², the leucine containing powder reduced influenzainfection by 2.9±0.2 log₁₀ TCID₅₀/mL, whereas the mannitol powder at acomparable dose (12.2 μg Ca^(2+/)cm²) reduced infection by 0.85±0.0log₁₀ TCID₅₀/mL and the maltodextrin powder (11.9 μg Ca²⁺/cm²) had noeffect on replication (FIG. 12). Even at higher doses (>27 μg Ca²⁺/cm²),the maximal reduction for mannitol (1.9±0.50 log 10 TCID₅₀/mL) andmaltodextrin (2.2±0.14 log₁₀ TCID₅₀/mL) was less than that of theleucine powder. Of note, previous testing using powders comprised of100% leucine found no effect of the excipient alone on viralreplication. These data suggest that the nature of the excipient canimpact the efficacy of calcium containing formulations.

Example 13

This example demonstrates the efficacy of dry powder formulationscomprising calcium salt, calcium lactate, calcium sulfate or calciumcitrate powders with respect to treatment of influenza, parainfluenza orrhinovirus.

The Formulation I, Formulation II, and Formulation III powders wereproduced by spray drying utilizing a Mobile Minor spray dryer (Niro, GEAProcess Engineering Inc., Columbia, Md.). All solutions had a solidsconcentration of 10 g/L and were prepared with the components listed inTable 22. Leucine and calcium salt were dissolved in DI water, andleucine and sodium salt were separately dissolved in DI water with thetwo solutions maintained in separate vessels. Atomization of the liquidfeed was performed using a co-current two-fluid nozzle (Niro, GEAProcess Engineering Inc., Columbia, Md.). The liquid feed was fed usinggear pumps (Cole-Parmer Instrument Company, Vernon Hills, Ill.) into astatic mixer (Charles Ross & Son Company, Hauppauge, N.Y.) immediatelybefore introduction into the two-fluid nozzle. Nitrogen was used as thedrying gas and dry compressed air as the atomization gas feed to thetwo-fluid nozzle. The process gas inlet temperature was 282° C. andoutlet temperature was 98° C. with a liquid feedstock rate of 70 mL/min.The gas supplying the two-fluid atomizer was approximately 14.5 kg/hr.The pressure inside the drying chamber was at −2″ WC. Spray driedproduct was collected in a container from a filter device.

TABLE 22 Formulations used to evaluate efficacy against differentrespiratory viruses Ca:Na molar Manufac- Lot # Formulation Compositionratio turing 26-190-F Formulation I 10.0% leucine, 1:2 Niro 35.1%calcium chloride, 54.9% sodium citrate (Active with 12.7% calcium ion)65-003-F Formulation III 10.0% leucine, 1:2 Niro 39.6% calcium chloride,50.4% sodium sulfate (Active with 14.3% calcium ion) 65-009-FFormulation II 10.0% leucine, 1:2 Niro 58.6% calcium lactate, 31.4%sodium chloride (Active with 10.8% calcium ion)

A cell culture model of Influenza A/Panama/2007/99, human parainfluenzatype 3 (hPIV3) or Rhinovirus (Rv16) infection was used to evaluate theefficacy of dry powder formulations. This model has been described indetail previously (See, Example 10) and utilizes Calu-3 cells grown atair-liquid interface as a model of influenza infection of airwayepithelial cells. Calu-3 cells were exposed to dry powders using a drypowder sedimentation chamber. The amount of calcium ion (Ca2+) deliveredto each well was determined by HPLC using dry powder recovered from anempty well in the cell culture plate. The concentration of calciumdeposited in each study is shown in Table 23.

TABLE 23 Calcium Deposition Formulation I Formulation III Formulation II(μg Ca/cm²) (μg Ca/cm²) (μg Ca/cm²) Low Medium High Low Medium High LowMedium High Influenza 12.74 17.12 28.85 11.37 15.84 27.73 10.93 16.0126.61 Parainfluenza 10.58 16.19 25.04 12.26 15.71 25.32 11.03 16.8126.33 Rhinovirus 11.63 16.25 24.11 10.86 15.01 23.89 11.49 15.22 24.69

One hour after exposure, cells were infected with 10 μL of InfluenzaA/Panama/99/2007 at a multiplicity of infection of 0.1-0.01 (0.1-0.01virions per cell), human parainfluenza type 3 (hPIV3) at a multiplicityof infection of 0.1-0.01 (0.1-0.01 virions per cell), or 10 μL ofrhinovirus (Rv16) at a multiplicity of infection of 0.1-0.01 (0.1-0.01virions per cell). Four hours after dry powder treatment, the apicalsurfaces were washed to remove excess formulation and unattached virus,and cells were cultured for an additional 20 hours at 37° C. plus 5%CO₂. The next day (24 hours after infection) virus released onto theapical surface of infected cells was collected in culture media and theconcentration of virus in the apical wash was quantified by TCID₅₀ (50%Tissue Culture Infectious Dose) assay. The TCID₅₀ assay is a standardendpoint dilution assay that is used to quantify how much of a givenvirus is present in a sample. For each of the three powders, Calu-3cells were exposed to three different Ca²⁺ doses and the replication ofeach virus was assessed.

Influenza

In the influenza model, all three powders significantly reduce viraltiter to comparable levels at the highest dose tested: Formulation I,Formulation III, and Formulation II reduced viral titer up to 3.25,3.80, and 3.95 log₁₀ TCID₅₀/mL, respectively (FIG. 13A). It is importantto note that while at the highest dose tested these powders exhibitedsimilar activity against influenza, at lower doses the data suggests themost efficacious powder was Formulation II (comprised of leucine,calcium lactate and sodium chloride). Formulation II reduced viraltiters 3.70 and 3.75 log₁₀ TCID₅₀/mL at low and medium doses, whereaslow doses of Formulation I and Formulation III reduced viral titer 2.50and 2.95 log₁₀ TCID₅₀/mL, and mid doses of Formulation I and FormulationIII reduced viral titers 2.65 and 3.30 log₁₀ TCID₅₀/mL, respectively.

Parainfluenza

Formulation I, Formulation II, and Formulation III were tested over asimilar dose range against parainfluenza. The parainfluenza titer in theFormulation III treated cell cultures was comparable to the controlcells (FIG. 13B) at doses of calcium similar to those used in theinfluenza experiment, indicating that the calcium sulfate basedformulation may exhibit activity only against specific pathogens. Incontrast, Formulation I and Formulation II treatment resulted in a dosedependent reduction in parainfluenza infection. At high doses,Formulation I and Formulation II reduced infection by 2.70 and 4.10log₁₀ TCID₅₀/mL, respectively, compared to the control cells. Similarly,Formulation II exhibited greater efficacy than Formulation I at themiddle dose tested, however, neither formulation reduced infection atthe lowest dose tested (FIG. 13B; Table 25). Collectively, these datademonstrate that calcium based dry powder formulations effectivelyreduce the infectivity of parainfluenza. These effects are specific tocertain calcium salts and the efficacious dose ranges differsignificantly from that observed for influenza.

Rhinovirus

Influenza and parainfluenza are enveloped viruses. To test the broadspectrum activity of calcium dry powder formulations and extend thesefindings to nonenveloped viruses, the same powders were tested againstrhinovirus. All three formulations reduced rhinovirus to some extent,with the Formulation II powder demonstrating the greatest activity (FIG.13C). Formulation II treatment resulted in a significant, 2.80 log₁₀TCID₅₀/mL viral reduction at the highest dose tested. Low and mediumdoses of this powder reduced titer 1.15 and 2.10 log₁₀ TCID₅₀/mL,respectively, compared to control cells. Formulation I and FormulationIII treatment also reduced rhinovirus infection, albeit to a lesserextent than Formulation II. At the highest dose tested, Formulation Ireduced infection by 1.70 log₁₀ TCID₅₀/mL and Formulation III reducedinfection 1.60 log₁₀ TCID₅₀/mL. Together these results indicate thatcalcium based dry powder formulations can be broadly applied to diverseviral infections.

The above data suggests that by increasing the delivered dose of calciumdry powder formulations exhibit more activity than was previouslyobserved at lower doses. Influenza infection was reduced by all threepowders tested, although the calcium lactate based formulation(Formulation II) exhibited greater potency than the calcium sulfate(Formulation III) and calcium citrate (Formulation III) formulations.Additionally, across all three viral strains, Formulation II treatmentresulted in the greatest reduction in viral titer. At higher dosesFormulation I effectively reduced viral titer in all three viralstrains, but the effect was much more pronounced with influenza andparainfluenza, suggesting a difference in mechanism that may be relatedto viral strain specificity. Formulation III treatment was activeagainst parainfluenza, but exhibited better activity against bothinfluenza and rhinovirus, suggesting that the specific calciumcounterions may have some role in the optimal activity of theformulation.

Example 14 Calcium Lactate, Sodium Chloride, Maltodextrin Dry Powder

This example describes the preparation of dry powders using feedstock ofFormulation XIV: 10.0 weight percent maltodextrin, 58.6 weight percentcalcium lactate and 31.4 weight percent sodium chloride.

An aqueous phase was prepared for a batch process by dissolvingmaltodextrin in ultrapure water, then calcium lactate pentahydrate, andfinally sodium chloride. The solution was kept agitated throughout theprocess until the materials were completely dissolved in the water atroom temperature. For the maltodextrin and calcium lactate formulation,three batches (A, B & C) of feedstock were prepared and spray dried.Details on the liquid feedstock preparations for each of the threebatches are shown in Table 24, where the total solids concentration isreported as the total of the dissolved anhydrous material weights. Thesolutions or suspensions were then spray dried using a Büchi spraydryer. For each formulation, three batches (A, B & C) of feedstock wereprepared and spray dried. Batch A, B and C particles were prepared usingthe corresponding feedstocks on a Büchi Mini spray dryer with processconditions similar to those used to spray dry for Formulations I-B andI-C in Example 1, with the exception of the following processconditions. The liquid feedstock flow rate was set at 5.2 mL/min forFormulation XIV-A and Formulation XIV-B and 5.6 mL/min for FormulationXIV-C. The outlet temperature was about 90° C. to 98° C. for FormulationXIV-A, about 100° C. to for Formulation XIV-B and about 100° C. 106° C.for Formulation XIV-C.

TABLE 24 Summary of liquid feedstock preparations of three batches ofparticles for Formulation XIV. Formulation: XIV-A XIV-B XIV-C Liquidfeedstock mixing Batch Batch Batch mixed mixed mixed Total solidsconcentration 5 g/L 5 g/L 5 g/L Total solids 5 g 5 g 20 g Total volumewater 1.0 L 1.0 L 4.0 L Amount leucine in 1 L 0.5 g 0.5 g 0.5 g Amountsodium chloride in 1.55 g 1.55 g 1.55 g 1 L Amount calcium lactate 4.13g 4.13 g 4.13 g pentahydrate in 1 L

Some of the physical properties of the particles obtained in threeseparate batches (Formulation XIV-A, XIV-B, and XIV-C) are summarized inTable 25. In addition to the data provided in Table 25, further dataabout the dry particles prepared by feedstock formulation XIV-A issummarized as follows. The fine particle fraction (FPF) as measured by acollapsed 2-stage Andersen Cascade Impactor with gravimetric analysiswas on average 71.3% for FPF less than 5.6 microns and 47.5% for FPFless than 3.4 microns. The volume size was determined by laserdiffraction on the HELOS/RODOS sizing equipment and the average valuefor the volume median diameter (×50) at a pressure of 1 bar was 1.40microns. In addition, the powder displayed flowrate independent behavioras can be seen from the ratio of ×50 measured at 0.5 bar to ×50 measuredat 4.0 bar, which was 1.04. The value for 1/4 bar for these particleswas 1.00, demonstrating the that particles were highly dispersable.

TABLE 25 Summary of ACI-2 data for the three batches of particles forFormulation XIV. Formulation: XIV-A XIV-B XIV-C FPF less than 5.6 μm onACI-2 (%) 71.3 66.6 68.2 FPF less than 3.4 μm on ACI-2 (%) 47.5 44.848.7

Additional information relating to properties of the Formulation XIVpowder and/or particles prepared in this example are provided in theTables or graphs shown in FIGS. 1A-1F

Example 15 Dispersibility

This example demonstrates the dispersibility of dry powder formulationscomprising calcium lactate, calcium sulfate or calcium citrate powderswhen delivered from different dry powder inhalers over a range ofinhalation maneuvers and relative to a traditional micronized drugproduct similarly dispersed.

The dispersibility of various powder formulations was investigated bymeasuring the geometric particle size and the percentage of powderemitted from capsules when inhaling on dry powder inhalers with flowrates representative of patient use. The particle size distribution andweight change of the filled capsules were measured for multiple powderformulations as a function of flow rate, inhaled volume and fill weightin 2 passive dry powder inhalers.

Powder formulations were filled into size 3 HPMC capsules (CapsugelV-Caps) by hand with the fill weight measured gravimetrically using ananalytical balance (Mettler Tolerdo XS205). Fill weights of 25 and 35 mgwere filled for Formulation I (lot #26-190-F), 25, 60 and 75 mg forFormulation II (Lot#69-191-1), 25 and 40 mg for Formulation III (Lot#65-009-F), 10 mg for a spray dried leucine powder (lot#65-017-F) and 25mg of micronized albuterol sulfate (Cirrus lot#073-001-02-039A). Twocapsule based passive dry powder inhalers (RS-01 Model 7, Low resistancePlastiape S.p.A. and RS-01 Model 7, High resistance Plastiape S.p.A.)were used which had specific resistances of 0.020 and 0.036 kPa1/2/LPMwhich span the typical range of dry powder inhaler resistance. Flow rateand inhaled volume were set using a timer controlled solenoid valve withflow control valve (TPK2000, Copley Scientific). Capsules were placed inthe appropriate dry powder inhaler, punctured and the inhaler sealed tothe inlet of the laser diffraction particle sizer (Spraytec, Malvern).The steady air flow rate through the system was initiated using theTPK2000 and the particle size distribution was measured via the Spraytecat 1 kHz for the durations at least 2 seconds and up to the totalinhalation duration. Particle size distribution parameters calculatedincluded the volume median diameter (Dv50) and the geometric standarddeviation (GSD) and the fine particle fraction (FPF) of particles lessthan 5 micrometers in diameter. At the completion of the inhalationduration, the dry powder inhaler was opened, the capsule removed andre-weighed to calculate the mass of powder that had been emitted fromthe capsule during the inhalation duration. At each testing condition, 5replicate capsules were measured and the results of Dv50, FPF andcapsule emitted powder mass (CEPM) were averaged.

In order to relate the dispersion of powder at different flow rates,volumes, and from inhalers of different resistances, the energy requiredto perform the inhalation maneuver was calculated and the particle sizeand dose emission data plotted against the inhalation energy. Inhalationenergy was calculated as E=R²Q²V where E is the inhalation energy inJoules, R is the inhaler resistance in kPa^(1/2)/LPM, Q is the steadyflow rate in L/min and V is the inhaled air volume in L.

FIG. 14 shows the dose emitted from a capsule for Formulation II powderat 3 different capsule fill weights, using both the high resistance andlow resistance RS-01 dry powder inhalers. At each fill weight, steadyinhalations ranged from a maximum energy condition of 9.2 Joules whichwas equivalent to a flow rate of 60 L/min through the high resistanceinhaler (R=0.036 kPa¹″²/LPM) with a total volume of 2 L down to lowerenergies with reduced volumes down to 1 L, reduced flow rates down to 15L/min and inhaler resistance down to R=0.020 kPa^(1/2)/LPM. As can beseen from FIG. 14, the entire mass of powder filled into the capsuleempties out of the capsule in a single inhalation for all 3 fill weightsof 25, 60 and 75 mg of Formulation II at the highest energy conditiontested. For the 25 mg fill weight, greater than 80% of the fill weightempties on average for all inhalation conditions down to 0.16 Joules. At60 mg, the capsule dose emission drops below 80% of the fill weight at0.36 Joules. At a capsule fill weight of 75 mg, the capsule doseemission drops below 80% of the fill weight at 1.2 Joules.

Also shown in FIG. 14 are 2 fill weights of 25 mg and 40 mg of amicronized albuterol sulfate drug formulation which was jet milled to anaverage particle size of 1.8 micrometers, hand filled into size 3capsules and dispersed in the high resistance RS-01 inhaler. As can beseen for both the 25 and 40 mg fill weights, at an inhalation energy of9.2 Joules (steady inhalation of 60 L/min for 2 L) the average CEPM isabove 80% of the capsule fill weight (93% for the 25 mg fill weight and84% for the 40 mg fill weight). However, at all measured lower energies,the CEPM drops to below 10 mg (<30% of capsule fill weight) for bothfill weights and monotonically decreases with decreases in inhalationenergy.

FIG. 15 shows the particle size distribution of the Formulation IIpowders that are emitted from the inhalers characterized by the volumemedian diameter (Dv50) and plotted against the inhalation energyapplied. Consistent values of Dv50 at decreasing energy values indicatethat the powder is well dispersed since additional energy does notresult in additional deagglomeration of the emitted powder. The Dv50values are consistent for all three fill weights of 75, 60 and 25 mg atall high energy values, with the Dv50 remaining below 2 micrometers downto 0.51 Joules for all 3 fill weights (FIG. 15). Taking into accountthat at the 60 and 75 mg fill weights, inhalations in the 0.5 to 1.2Joule range did not fully emit the powder from the capsule (FIG. 14), itis clear that the powder which was emitted was still fully dispersed bythe DPI (FIG. 15). In this range, the Dv50 is not significantlyincreased in size, which would be expected if the emitting powdercontained a lot of agglomerates and was not well dispersed.

Also shown in the FIG. 15 are fill weights of 25 mg(×) and 40 mg(+) of amicronized albuterol sulfate drug formulation which was jet milled to anaverage particle size of 1.8 micrometers, hand filled into size 3capsules and dispersed in the high resistance RS-01 inhaler. As can beseen for both the 25 and 40 mg fill weights, at an inhalation energy of9.2 Joules (steady inhalation of 60 L/min for 2 L) the average Dv50 isbelow 2 micrometers (1.8 and 1.6 μm respectively) for both fill weights,demonstrating good dispersion and relatively few agglomerates. However,at all measured lower energies, the Dv50 increases to greater than 2micrometers (3.9 and 3.1 μm respectively) and continues to monotonicallyincrease with decreasing inhalation energy, demonstrating agglomerationand poor dispersion of the primary particles.

Additional powders were tested at all of the test conditions and averageCEPM and Dv50 were determined (Table 26) These results demonstrate theability of the powder formulations to be fully emptied anddeagglomerated at inhalation energies down to approximately 0.5 Joules.

TABLE 26 Mean CEPM, Dv(50) and FPF as a function of fill weight,flowrate and duration for FORMUALTIONS I-III and placebo. InhalationFill Flow Energy, Mean Mean Mean Weight Rate Duration E = R²Q²V CEPMDv(50) FPF, Powder DPI (mg) (LPM) (s) (Joules) (mg) (μm) % <5 μmFormulation I RS.01.HR 25 15 4 0.29 15.84 4.77 52.09 Formulation IRS.01.HR 25 20 3 0.51 22.88 3.46 65.79 Formulation I RS.01.HR 25 30 21.15 24.75 2.94 72.88 Formulation I RS.01.HR 25 60 2 9.18 24.72 2.9373.39 Formulation I RS.01.LR 25 15 4 0.09 4.30 7.29 31.97 Formulation IRS.01.LR 25 20 3 0.16 8.05 5.10 48.98 Formulation I RS.01.LR 25 30 20.36 19.94 3.28 71.09 Formulation I RS.01.LR 25 60 2 2.85 24.75 2.5180.26 Formulation I RS.01.HR 35 30 2 1.15 33.77 2.17 83.17 Formulation IRS.01.HR 35 60 2 9.18 34.73 2.33 81.42 Formulation I RS.01.LR 35 30 20.36 13.07 3.16 73.22 Formulation I RS.01.LR 35 60 2 2.85 34.57 2.3483.15 Placebo RS.01.HR 10 15 4 0.29 3.87 25.71 6.22 Placebo RS.01.HR 1020 3 0.51 8.79 22.80 8.64 Placebo RS.01.HR 10 30 2 1.15 9.42 22.95 11.83Placebo RS.01.HR 10 60 2 9.18 9.78 21.45 12.52 Placebo RS.01.LR 10 15 40.09 1.87 40.36 3.17 Placebo RS.01.LR 10 20 3 0.16 3.08 28.16 5.20Placebo RS.01.LR 10 30 2 0.36 7.01 18.62 9.39 Placebo RS.01.LR 10 60 22.85 9.82 15.26 16.41 Formulation III RS.01.HR 25 15 4 0.29 24.87 3.2668.77 Formulation III RS.01.HR 25 20 3 0.51 25.48 3.06 72.61 FormulationIII RS.01.HR 25 30 2 1.15 25.05 2.90 74.06 Formulation III RS.01.HR 2560 2 9.18 25.28 2.92 71.87 Formulation III RS.01.LR 25 15 4 0.09 18.975.59 43.81 Formulation III RS.01.LR 25 20 3 0.16 24.95 3.45 68.14Formulation III RS.01.LR 25 30 2 0.36 25.08 2.72 76.82 Formulation IIIRS.01.LR 25 60 2 2.85 24.88 2.66 75.76 Formulation III RS.01.HR 40 30 21.15 39.55 2.76 74.92 Formulation III RS.01.HR 40 60 2 9.18 40.13 3.1467.35 Formulation III RS.01.LR 40 30 2 0.36 39.74 2.89 75.51 FormulationIII RS.01.LR 40 60 2 2.85 39.85 2.65 77.00 Formulation II RS.01.HR 25 154 0.29 24.45 3.56 63.96 Formulation II RS.01.HR 25 17.5 3.4 0.39 21.432.34 80.07 Formulation II RS.01.HR 25 20 3 0.51 23.55 2.15 82.08Formulation II RS.01.HR 25 25 2.4 0.80 24.42 1.39 90.70 Formulation IIRS.01.HR 25 30 2 1.15 24.88 1.28 88.29 Formulation II RS.01.HR 25 60 29.18 25.07 1.59 85.28 Formulation II RS.01.LR 25 15 4 0.09 7.47 7.4632.20 Formulation II RS.01.LR 25 20 3 0.16 20.39 4.29 57.09 FormulationII RS.01.LR 25 30 2 0.36 24.23 2.52 78.85 Formulation II RS.01.LR 25 602 2.85 24.81 1.61 89.78 Formulation II RS.01.HR 60 25 2.4 0.80 52.420.99 90.45 Formulation II RS.01.HR 60 30 2 1.15 56.50 0.78 92.70Formulation II RS.01.HR 60 60 2 9.18 59.42 1.19 90.64 Formulation IIRS.01.LR 60 30 2 0.36 26.62 2.48 80.08 Formulation II RS.01.LR 60 60 22.85 59.51 1.19 90.64 Formulation II RS.01.HR 75 25 2.4 0.80 47.63 1.3689.83 Formulation II RS.01.HR 75 30 2 1.15 51.84 1.07 92.59 FormulationII RS.01.HR 75 60 2 9.18 74.90 1.41 85.20 Micronized Albuterol073-001-02- RS.01.HR 25 15 4 0.29 3.12 16.76 13.00 039A MicronizedAlbuterol 073-001-02- RS.01.HR 25 20 3 0.51 5.00 8.40 32.10 039AMicronized Albuterol 073-001-02- RS.01.HR 25 30 2 1.15 7.08 3.86 59.44039A Micronized Albuterol 073-001-02- RS.01.LR 25 60 2 2.85 15.28 2.5775.01 039A Micronized Albuterol 073-001-02- RS.01.HR 25 60 2 9.18 23.181.77 81.65 039A Micronized Albuterol 073-001-02- RS.01.HR 40 15 4 0.292.43 17.63 10.73 039A Micronized Albuterol 073-001-02- RS.01.HR 40 20 30.51 4.97 6.34 42.24 039A Micronized Albuterol 073-001-02- RS.01.HR 4030 2 1.15 8.55 3.13 67.18 039A Micronized Albuterol 073-001-02- RS.01.LR40 60 2 2.85 18.88 2.62 73.98 039A Micronized Albuterol 073-001-02-RS.01.HR 40 60 2 9.18 33.40 1.60 84.30 039A

Example 16 Solid State Particle Analysis

A. X-Ray Powder Diffraction

Formulations I, II, III and XIV were analyzed for amorphous/crystallinecontent and polymorphic form using high resolution X-ray powderdiffraction (XRPD) and differential scanning calorimetry (DSC). ForXRPD, phase identification was performed to identify any crystallinephases observed in each XRPD pattern. XRPD patterns were collected usinga PANalytical X'Pert Pro diffractometer (Almelo, The Netherlands). Thespecimen was analyzed using Cu radiation produced using an Optix longfine-focus source. An elliptically graded multilayer minor was used tofocus the Cu Kα X-rays of the source through the specimen and onto thedetector. The specimen was sandwiched between 3-micron thick films,analyzed in transmission geometry, and rotated to optimize orientationstatistics. A beam-stop was used, along with helium purge in some cases,to minimize the background generated by air scattering. Soller slitswere used for the incident and diffracted beams to minimize axialdivergence. Diffraction patterns were collected using a scanningposition-sensitive detector (X'Celerator) located 240 mm from thespecimen. The data-acquisition parameters of each diffraction patternare displayed above the image of each pattern in appendix C. Prior tothe analysis a silicon specimen (NIST standard reference material 640c)was analyzed to verify the position of the silicon 111 peak. Calculatedpatterns for the potential crystalline components (including anhydrousand hydrated forms) were produced from either the Cambridge StructuralDatabase or the International Center for Diffraction Data (ICDD)Database and compared to the experimental patterns. The crystallinecomponents were qualitatively determined XRPD was also performed onpowders that had been conditioned at 75% RH for a period of three tofour hours in a Dynamic Vapor Sorption system in order to assess thepropensity for recrystallization of said powders upon short-termexposure to elevated humidities.

Differential scanning calorimetry (DSC) was performed using a TAInstruments differential scanning calorimeter Q2000 (New Castle, Del.).The sample was placed into an aluminum DSC pan, and the weightaccurately recorded. The data acquisition and processing parameters aredisplayed on each thermogram. Indium metal was used as the calibrationstandard. The glass transition temperature (T_(g)) is reported from theinflection point of the transition/or/the half-height of the transition.Standard mode DSC experiments were initially conducted on the powders ofinterest in order to assess the overall thermal behavior of the powders.Cyclic mode DSC experiments were also performed in order to attempt toidentify the occurrence of glass transitions occurring in these powdersover temperature regions of interest identified in the standard DSCthermograms.

Surprisingly, high calcium and sodium salt content powders were producedthat possessed a mixture of amorphous and crystalline content thatpossessed optimized properties with respect to their dispersibility andstability in the dry state and their dissolution and water absorptionproperties in the hydrated state. As shown in FIGS. 16 and 17, theFormulation I powder was observed via XRPD to consist of a combinationof crystalline sodium chloride and a poorly crystalline or amorphouscalcium citrate and potentially calcium chloride-rich phase (asevidenced by a lack of observance of any characteristic peaks for anycalcium salt forms in this powder as well as the absence of anycharacteristic peaks for leucine). As shown in FIG. 18, a glasstransition temperature of approximately 167° C. was observed via cyclicDSC for the amorphous calcium-rich phase, indicating that this amorphousphase should be relatively stable to crystalline conversion at standardconditions (25° C., 30% RH). The presence of crystalline sodium chloridein this powder in the dry state may enhance the dispersibility andstability of said powder. The presence of the calcium salt in a poorlycrystalline or amorphous form in the Formulation I powder may alsofacilitate the rapid water uptake and dissolution properties of theFormulation I formulation upon deposition in the lungs (i.e.,crystalline sodium chloride is readily soluble, whereas calcium citrateis poorly soluble).

Similar results were seen for powders Formulation II and FormulationXIV. As shown in FIGS. 19 and 20, the Formulation II powder was observedvia XRPD to consist of a combination of crystalline sodium chloride anda poorly crystalline or amorphous calcium lactate and potentiallycalcium chloride-rich phase (as evidenced by a lack of observance of anycharacteristic peaks for any calcium salt forms in this powder as wellas the absence of any characteristic peaks for leucine). As shown inFIG. 21, a glass transition temperature of approximately 144° C. wasobserved via cyclic DSC for the amorphous calcium-rich phase, indicatingthat this amorphous phase should be relatively stable to crystallineconversion at standard conditions (25° C., 30% RH). Nearly identicalresults were seen for the Formulation XIV powder which contained 10%maltodextrin versus 10% leucine (see FIGS. 22 and 23) for XRPD data aswell as FIG. 24 which shows a glass transition temperature ofapproximately 134° C.

In contrast, the Formulation III formulation displayed the presence ofsome degree of crystalline calcium salt content (calcium sulfate) inaddition to crystalline sodium chloride (see FIGS. 25A and 25B).However, this powder still possessed a significant degree of amorphous,calcium-rich phase content, as evidenced by the presence of a glasstransition temperature of approximately 159° C. via DSC (see FIG. 26).

B. Surface RAMAN Mapping

Surface Mapping RAMAN experiments were conducted on samples ofFormulations I through III and XIV in order to determine the nature ofthe chemical composition at the surface of the particles comprisingthese formulations. Raman map spectra were acquired on a Renishaw inViaRamascope (Gloucestershire, UK) equipped with a Leica DM LM microscope(Wetzlar, Germany). The instrument was calibrated using a silicon waferstandard. The samples were prepared for analysis on an aluminum-coatedmicroscope slide. The excitation wavelength was 785 nm using ahigh-power near-infrared diode laser source. The data collection forFormulation I, Formulation III and Formulation XIV was a static scanwith a 30 second exposure time and 10 accumulations. The data collectionfor Formulation II was an extended scan with a 60 second exposure timeand one accumulation. A Philips ToUcam Pro II camera (model PCVC 840K)(Amsterdam, the Netherlands) was used for image acquisition with a 50×objective. Renishaw WiRE 3.1 (service pack 9) software (Gloucestershire,UK) was used for data collection and processing.

Raman spectra were acquired for six particles from the Formulation Isample, and are shown overlaid in FIG. 27A. Spectra files 389575-1 and389575-6 are characterized by the presence of weak peaks atapproximately 1450, 965 and 850 cm-1. These peaks are discernable asonly very weak features in spectra file 389575-6, and are not detectedin the remaining spectral data files. In FIG. 27B, spectrum 389575-6 isbackground subtracted and overlaid with the Raman spectra of calciumcitrate tetrahydrate, sodium citrate, and leucine. The sample spectrumexhibits peaks at approximately 1450 and 850 cm-1 which are common toboth leucine and the citrate salts. The sample spectrum displays anadditional peak at approximately 965 cm-1, which is consistent with therelatively stronger intensity peak in the spectrum of the citrate salts(i.e., calcium citrate tetrahydrate and sodium citrate). Thecharacteristic leucine peak at 1340 cm-1 is not observed in the samplespectra.

Raman spectra were acquired for eight particles from the Formulation IIIsample, and are shown overlaid in FIG. 27C. All particle spectra arecharacterized by the presence of a peak at approximately 1060 cm-1. Anadditional peak at approximately 670 cm-1 is observed in spectral file388369-4. The 670 cm-1 peak is also observable in spectral data files388369-1, 3, and 8 after background subtraction (not shown). In FIG.27D, spectrum 388369-4 is background subtracted and overlaid with theRaman spectra of calcium sulfate, calcium sulfate dihydrate, sodiumsulfate anhydrous, and leucine. The background subtracted samplespectrum reveals a possible third peak near 520 cm-1. The peaks at 1060and 670 cm-1 are present at similar positions to characteristic peaks ofthe sulfate ions displayed, but do not overlap precisely. Thefrequencies of the peaks at 1060 and 670 cm-1 in the sample spectrum areconsistent with the stretching and bending modes, respectively, of asulfate ion functional group. Peaks assignable to leucine are notdetected in the particle spectra.

Raman spectra were acquired for twelve particles from the Formulation IIsample, and are shown overlaid in FIG. 27E. All particle spectra arecharacterized by the presence of peaks at approximately 1045 and 860cm-1. Additional peaks can be observed in various spectra atapproximately 1450, 1435, 1125, 1095, 930, and 775 cm-1, which generallycorrelate in relatively intensity with the strong peak at 1045 cm-1. InFIG. 27F, spectra 389576-7 and 389576-12 are background subtracted andoverlaid with the Raman spectra of calcium lactate pentahydrate, andleucine. A good correspondence is observed between the sample spectraand calcium lactate pentahydrate spectrum. However, the sample spectradisplay additional weak peaks at approximately 1345, 1170, 960, 830, and760 cm-1 which are absent in the spectrum of calcium lactatepentahydrate. Similar peaks are present in the reference spectrum ofleucine, although with slightly different relative intensities andfrequencies.

Raman spectra were acquired for twelve particles from the FormulationXIV sample, and are shown overlaid in FIG. 27G. All particle spectra arecharacterized by the presence of a peak at approximately 1045 cm-1. Allparticle spectra except file 389577-2 also display a peak atapproximately 860 cm-1. Additional peaks can be observed in variousspectra at approximately 1450, 1435, 1125, 1095, 930, and 775 cm-1,which generally correlate in relatively intensity with the strong peakat 1045 cm-1. In FIG. 27H, spectrum 389577-9 is background subtractedand overlaid with the Raman spectra of calcium lactate pentahydrate. Agood correspondence is observed between the sample and calcium lactatepentahydrate spectra. Peaks assigned to maltodextrin (not shown) are notobserved in the sample spectra.

Thus, RAMAN surface mapping analysis indicates that the surfacecomposition of each of Formulations I though XIV is dominated by thepresence of the various calcium salts (calcium citrate for FormulationI, calcium sulfate for Formulation III and calcium lactate forFormulations II and XIV). For the case of Formulations I through III,this is in contrast to the reported use of leucine as adispersion-enhancing agent that increases the dispersibility of powdersfor aerosolization via being concentrated at the surface of theparticles comprising said powders. For the formulations disclosedherein, it does not appear that leucine is acting as a dispersibilityenhancer in this fashion, as also evidenced by the similar results seenfor Formulations II (leucine-containing calcium lactate formulation) andXIV (maltodextrin-containing calcium lactate formulation) with respectto surface content and dispersibility.

Example 17 Ion Exchange Reaction for Spray Drying Supersaturated CalciumCitrate and Calcium Sulfate

Saturated or super-saturated stocks of aqueous calcium sulfate orcalcium citrate were prepared for spray drying using calcium chlorideand sodium sulfate or calcium chloride or calcium citrate as startingmaterials. A range of total solids concentrations from 5 to 30 g/L wereprepared both by (i) pre-mixing both salts in water and (ii) keeping thecalcium and sodium salt in separate aqueous solutions, with staticmixing in-line immediately before spray drying. All of the liquid feedstocks prepared contained saturated or supersaturated calcium sulfateamounts, (where the solubility limit of calcium sulfate in water is 2.98g/L) and saturated or supersaturated calcium citrate amounts (where thesolubility limit of calcium citrate in water is 0.96 g/L). Consideringthe calcium chloride and sodium sulfate precipitation reaction proceedsto completion (CaCl₂+Na₂SO4→CaSO₄+2 NaCl), the corresponding finalconcentrations of calcium sulfate are listed in Table 24. Similarresults for the calcium chloride and sodium citrate precipitationreaction (3CaCl₂+2Na₃C₆H₅O₇→Ca₃(C₆H₅O₇)₂+6NaCl) are also shown in Table28.

TABLE 28 Liquid feedstock total solids concentrations and final calciumsulfate or calcium citrate concentrations, where the aqueous solubilitylimit of calcium sulfate is 2.98 g/L and calcium citrate is 0.96 g/LTotal solids Final calcium sulfate Final calcium citrate concentrationconcentration concentration (g/L) (g/L) (g/L) 5 2.7 2.9 10 5.4 5.9 158.1 8.8 20 10.8 11.7 30 16.1 17.6

Formulations of 44 weight percent calcium chloride and 56 weight percentsodium sulfate were produced by spray drying utilizing a Mobile Minorspray dryer (Niro, GEA Process Engineering Inc., Columbia, Md.). Theliquid feed stocks were prepared at a range of solids concentration from5-30 g/L. For pre-mixed feeds, sodium salt then calcium salt wasdissolved in DI water with constant stirring on a magnetic stirplate.For static mixed feeds, calcium salt was dissolved in DI water, andsodium salt was separately dissolved in DI water with the two solutionsmaintained in separate vessels with constant agitation. Atomization ofthe liquid feed was performed using a co-current two-fluid nozzle (Niro,GEA Process Engineering Inc., Columbia, Md.). The liquid feed was fedusing gear pumps (Cole-Panner Instrument Company, Vernon Hills, Ill.)either directly into the two-fluid nozzle for pre-mixed feeds or into astatic mixer (Charles Ross & Son Company, Hauppauge, N.Y.) immediatelybefore introduction into the two-fluid nozzle for static mixed feeds.Nitrogen was used as the drying gas and dry compressed air as theatomization gas feed to the two-fluid nozzle. The process gas inlettemperature was 240-250° C. and outlet temperature was 94-988° C. with aliquid feedstock rate of 50-70 mL/min. The gas supplying the two-fluidatomizer was approximately 11 kg/hr. The pressure inside the dryingchamber was at −2″ WC. Spray dried product was collected from a cycloneand analyzed for volume particle size by laser diffraction using a HELOSwith RODOS attachment and for aerosol properties using a collapsedtwo-stage ACI.

Pre-mixed feeds were assessed for solution stability and clarity. At atotal solids concentration of 5 g/L, where the final calcium sulfateconcentration would be slightly over the solubility limit of calciumsulfate, the solution stayed clear during the 30 minute duration ofmixing and spray drying. As the total solids concentration increased andthe final calcium sulfate concentration greatly exceeded the solubilitylimit, the feed stock became cloudy and precipitation was evident. At 10g/L the liquid was slightly cloudy, at 20 g/L the liquid was clear forapproximately 5-10 minutes before becoming increasingly cloudy over thecourse of 10 minutes and at 30 g/L the liquid was clear forapproximately 2 minutes after mixing, with visible precipitationappearing after approximately 5 minutes.

The pre-mixed and static mixed liquid feed stocks were spray dried andthe resulting dry powder collected from the cyclone. Results from theHELOS with RODOS are shown in FIG. 28 with representative particle sizedistributions shown in FIG. 29. While an increase in particle size isexpected with increasing feed stock solids concentrations (as seen inthe static mixed feeds), the significant particle size increase andbroadened particle size distribution in the pre-mixed feeds isundesirable.

Results for aerosol characterization of the dry powders using thecollapsed ACI are shown in FIG. 30.

Unstable solutions with continued precipitation may negatively affectreproducible particle formation during spray drying and also result in abroad particle size distribution. The supersaturated, clear solutionsevident for 2-10 minutes for the higher solids concentration suggestthat the solutions could be static mixed to achieve a higher spraydrying throughput while reproducibly producing a narrow particle sizedistribution.

Similar results were exemplified for calcium citrate, as demonstrated inExample 1 for the formulation comprising 10.0 weight percent leucine,35.1 weight percent calcium chloride and 54.9 weight percent sodiumcitrate (Formulation I-A). The precipitation reaction will result in aformulation comprising 10.0 weight percent leucine, 52.8 weight percentcalcium citrate and 37.2 weight percent sodium chloride. At a totalsolids concentration of 10 g/L, the final calcium citrate concentrationwould be 5.3 g/L, which exceeds the solubility limit of calcium citratein water of 0.96 g/L. As can be seen from the properties of the spraydried powder (FIGS. 1A-1E and 2-4), this supersaturated solutionresulted in respirable particles with narrow size distribution.

Example 18

Small, dispersible particles were made from calcium-containingformulations with and without leucine, as well as magnesium-containingand sodium only formulations.

The following powders were spray dried on the Mali B-290 using the highperformance cyclone with an air feed rate of 30 mm air, aspirator at 90%rate and the small glass collection vessel. The inlet temperature was220° C. and the outlet temperature was between 96-102° C. The solidsconcentration was 5 g/L and all were mixed in D.I. water by fullydissolving one component at a time, before adding the next in the orderin which they are listed.

-   -   18-1) 10.0% lactose, 30.6% magnesium chloride, 59.4% sodium        citrate, Ca:Na ratio=1:2    -   18-2) 63.4% magnesium lactate, 36.6% sodium chloride, Ca:Na        ratio=1:2    -   18-3) 10.0% leucine, 58.4% magnesium lactate, 31.6% sodium        chloride, Ca:Na ratio=1:2    -   18-4) 50.0% leucine, 50% calcium lactate    -   18-5) 10% leucine, 90% sodium chloride    -   18-6) 60% leucine, 40% sodium chloride    -   18-7) 10.0% albuterol, 58.6% calcium lactate, 31.4% sodium        chloride    -   18-8) 90.0% albuterol, 5.9% calcium lactate, 3.1% sodium        chloride

Characterization results for these powders are shown in Table 29 below.All eight powders exhibited good dispersibility with respect to ×500.5/4 and 1/4 ratios. FPF's<5.6 microns ranged from a low of 18.7% to75.6%.

TABLE 29 Assorted sodium, calcium and magnesium-based formulations.FPF_TD FPF_TD x50 (μm) GSD @ <3.4 um <5.6 % Mass Lot Formulation Method@ 1 bar 1 bar 1/4 bar 0.5/4 bar % um % collected yield % 68.124.1lact:MgCl2:Na3Cit Buchi HP 2.9 2.3 1.1 1.1 18.1% 37.8% 55.7% 88.9%10:30.6:59.4 68.129.1 leucine:MgLact:NaCl Buchi HP 2.7 2.4 0.8 1.1 14.5%32.3% 53.0% 80.0% 10:58.6:31.4 68.129.2 MgLact:NaCl 63.4:36.6 Buchi HP3.3 2.1 1.0 1.0 16.5% 39.3% 59.8% 78.0% 68.125.1 leu:CaLact 50:50 BuchiHP 3.5 2.2 1.1 1.1 19.2% 38.5% 60.4% 76.0% 68.124.2 leu:NaCl 10:90 BuchiHP 1.1 1.7 1.0 1.2 53.0% 71.0% 78.6% 67.9% 68.124.3 leu:NaCl 60:40 BuchiHP 1.4 2.2 1.1 1.2 49.7% 75.6% 85.2% 54.3% 68.125.2albuterol:CaLact:NaCl Buchi HP 2.8 2.3 0.9 1.0 16.0% 38.6% 60.2% 81.5%10:58.6:31.4 68.125.3 albuterol:CaLact:NaCl Buchi HP 3.5 2.3 1.0 1.18.9% 18.7% 29.1% 40.5% 90:5.9:3.1

Several additional calcium-free exemplary formulations were producedutilizing various spray-dryer systems (Buchi, Labplant and Niro-basedsystems) following similar procedures those described above. Selectedcharacterization results for the resultant powders are shown in Table 30(cells with blank values indicates no value was measured for thatpowder).

TABLE 30 Non-calcium formulations of small, dispersible powders FPF_TDFPF_TD x50 (μm) GSD @ <3.4 um <5.6 um % Mass Lot Formulation Method @ 1bar 1 bar 1/4 bar 0.5/4 bar water % % % collected yield % NaCl 2.26.2NaCl, 100 Labplant 2.9 1.4 0.5% 27.115.4 NaCl 100 Niro 4.5 1.9 1.4 0.6%5.2% 22.0% 43.1% 61.3% Magnesium Salts 27.33.2 MgCl2 + NaCl Labplant 4.31.9 1.2 29.9% 2.3% 5.7% 14.0% 17.9% 27.15.4 MgCl2:Na2CO3, 47:53 Labplant2.3 1.4 1.1 87.4% 17.6% 68.124.1 lactose:MgCl2:Na3Cit Buchi HP 2.9 2.31.1 1.1 18.1% 37.8% 55.7% 88.9% 10:30.6:59.4 68.129.1leucine:MgLact:NaCl Buchi HP 2.7 2.4 0.8 1.1 14.5% 32.3% 53.0% 80.0%10:58.6:31.4 68.129.2 MgLact:NaCl Buchi HP 3.3 2.1 1.0 1.0 16.5% 39.3%59.8% 78.0% 63.4:36.6 Leucine 26.155.1 Leucine, 100 Buchi HP 4.1 2.3 1.133.6% 58.5% 71.8% 56.7%

Further, several additional examples of compositions containing eitherno excipients or non-leucine excipients were also produced utilizingvarious spray-dryer systems (Buchi, Labplant and Niro-based systems)following similar procedures those described above. Selectedcharacterization results for the resultant powders are shown in Table 31(cells with blank values indicates no value was measured for thatpowder).

TABLE 31 Non-leucine salt formulations of small, dispersible powdersFPF_TD FPF_TD x50 (μm) GSD @ 1/4 0.5/4 <3.4 um <5.6 um % Mass LotFormulation Method @ 1 bar 1 bar bar bar water % % % collected yield %Excipients with lactate 45.132.1 leu:mdextrin:CaLact:NaCl Buchi HP 1.51.9 1.0 1.0 31.8% 53.7% 62.9% 65.6% 5:5:58.6:31.4 45.137.1lact:CaLact:NaCl 10:58.6:31.4 Buchi HP 2.7 2.0 1.0 1.0   8% 24.9% 48.1%63.4% 81.4% 45.137.2 mannitol:CaLact:NaCl 10:58.6:31.4 Buchi HP 1.5   6%43.6% 66.6% 73.1% 68.6% 45.189.2 mannitol:CaLact:NaCl 10:58.6:31.4 BuchiHP 1.2 1.8 1.0 1.0   5% 44.8% 66.0% 71.6% 45.137.3 mdextrin:CaLact:NaCl10:58.6:31.4 Buchi HP 1.4 1.9 1.0 1.0   6% 47.5% 71.3% 77.6% 77.7%45.189.3 mdextrin:CaLact:NaCl 10:58.6:31.4 Buchi HP 1.3 1.8 1.0 1.0   7%44.8% 66.6% 73.2% 45.137.4 trehalose:CaLact:NaCl 10:58.6:31.4 Buchi HP1.4 1.9 1.0 1.0   4% 51.3% 72.8% 78.2% 77.2% Calcium Citrate 2.26.3CaCl2:Na3Cit 39:61 Labplant 3.3 1.2 1.0 11.0%  22.8% 26.048.2CaCl2:Na3Cit2 39:61 Niro 7.0 2.1 1.2 7.9% 22.0% 46.1% 61.0% 27.03.1CaCl2:Na3Cit 39:61 Labplant 3.6 1.4 1.1 9.0% 25.1% 26.013.3 CaCl2:Na3Cit49:51 not to Niro 3.6 2.0 1.1 12.7% 31.0% 45.9% 43.9% completion27.183.4 Ca(OH)2:Cit acid:NaCl 35:61:3.5 Buchi 2.6 1.8 1.0 9.3% 17.7%21.5% 23.1% Calcium Sulfate 2.26.4 CaCl2:Na2SO4 44:56 Labplant 3.7 1.71.4 5.1% 12.1% 26.060.1 CaCl2:Na2SO4 44:56 Niro 3.0 2.0 1.3 15.3% 40.2%62.9% 60.8% 26.060.3 CaCl2:Na2SO4 44:56-static mixed Niro 2.6 1.6 1.217.0% 42.5% 58.6% 31.4% 26.069.1 CaCl2:NaSO2 44:56 5 g/L Niro 2.9 1.61.4 11.1% 38.5% 59.1% 25.2% 26.069.2 CaCl2:NaSO2 44:56 10 g/L Niro 3.51.8 1.5 7.6% 27.7% 61.1% 45.6% 26.069.3 CaCl2:NaSO2 44:56 20 g/L Niro4.0 2.1 1.4 6.9% 25.3% 62.6% 37.3% 26.124.1 CaCl2:Na2SO4, 44:56 5 g/LNiro 2.9 1.5 1.5 6.5% 11.0% 34.5% 53.4% 22.0% 26.124.2 CaCl2:Na2SO4,44:56 10 g/L Niro 3.2 1.5 1.7 7.1% 9.9% 28.9% 45.1% 35.0% 27.114.5CaCl2:Na2SO4 44:56 Niro 4.1 1.8 1.6 6.8% 5.8% 22.6% 50.2% 52.5% 27.154.1CaCl2:Na2SO4 44:56 Buchi 3.1 1.9 1.3 14.0% 31.6% 55.1% 50.3% 27.114.6CaCl2:Na2SO4:Rhod B 44:56:1 Niro 3.9 1.9 1.0 7.2% 7.4% 25.5% 52.4% 44.2%27.114.1 lact:CaCl2:Na2SO4 90:4.4:5.6 Niro 3.9 2.5 1.2 17.9%  12.0%28.5% 42.5% 13.3% 27.114.2 lact:CaCl2:Na2SO4 50:22:28 Niro 4.5 2.0 1.112.6%  10.2% 29.1% 44.5% 58.0% 27.115.3 CaSO4 100 Niro 3.8 1.7 1.214.0%  15.8% 38.2% 57.0% 47.5% 27.185.2 Ca(OH)2:Sulf acid:NaCl Buchi 2.51.8 1.3 17.5% 45.2% 65.2% 44.1% 41.3:54.6:4.1 27.185.3 Ca(OH)2:Sulf acid43:57 Buchi 2.9 2.3 1.1 15.3% 38.9% 59.4% 16.1% 27.183.1 CaLact:NaCl96.8:3.2 Buchi 3.1 2.0 1.1 22.4% 50.9% 69.5% 35.0% 27.115.2 CaCl2:Na2CO351:49 Niro 3.9 2.1 1.4 1.7% 8.4% 22.4% 38.9% 27.3% 27.184.3 CaGluc:NaCl98.3:1.7 Buchi 2.9 2.0 1.0 13.5% 26.7% 48.3% 47.6% 27.15.2 MgCl2:Na3Cit,36:64 Labplant 3.1 1.4 1.0 13.2%  28.6% 27.33.3 MgCl2:Na3Cit, 36:64Labplant 4.0 2.2 1.2 15.7%  21.4% 53.7% 68.2% 26.2% 27.15.3MgCl2:Na2SO4, 40:60 Labplant 3.9 2.3 1.3 11.1%  31.8% 27.33.9MgCl2:Na2CO3, 47:53 Labplant 2.7 3.7 1.4 7.9% 21.0% 46.0% 58.3% 18.8%27.15.4 MgCl2:Na2CO3, 47:53 Labplant 2.3 1.4 1.1 87.4%  17.6% 68.124.1lact:MgCl2:Na3Cit 10:30.6:59.4 Buchi HP 18.1% 37.8% 55.7% 88.9% 68.129.2MgLact:NaCl 63.4:36.6 Buchi HP 16.5% 39.3% 59.8% 78.0%

Table 32 contains characterization data for additional leucine andcalcium containing small and dispersible powder compositions made viausing a Buchi or a Niro spray-drying system per procedures similar tothose described above (cells with blank values indicates no value wasmeasured for that powder).

TABLE 32 Leucine and calcium-containing formulations of small,dispersible particles x50 FPF_TD FPF_TD Tapped (μm) @ GSD @ 1/4 <3.4 um<5.6 um % Mass density Lot Formulation Method 1 bar 1 bar bar 0.5/4 barwater % % % collected yield % (g/cc) Chloride 26.010.2 leu:CaCl2:NaClNiro 4.8 2.2 1.1 15.8% 35.9% 50.8% 64.1% 50:29.5:20.5 26.041.3leu:CaCl2:NaCl Niro 4.9 2.4 14.7% 28.0% 43.0% 50.2% 50:29.5:20.5 Citrate26.013.1 leu:CaCl2:Na3Cit2 Niro 4.2 2.1 1.6 16.8% 35.2% 53.8% 56.1%50:19.5:30.5 26.013.2 leu:CaCl2:Na3Cit2 Niro 4.8 1.8 1.3 20.8% 39.6%52.2% 57.5% 10:35.1:54.9 26-190-F Leucine:CaCl2:Na3Cit2 Niro 2.6 1.9 1.21.2 45.7% 61.6% 66.3% 74.8% 0.29 10.0:35.1:54.9 Sulfate 26.013.4leu:CaCl2:Na2SO4 Niro 3.7 2.0 1.4 19.6% 39.4% 60.9% 73.1% 10:39.6:50.426.060.2 leu:CaCl2:Na2SO4 Niro 2.9 1.9 1.2 16.2% 35.2% 53.2% 46.5% 0.1810:39.6:50.4 26.060.4 leu:CaCl2:Na2SO4 Niro 2.9 1.7 1.3 18.8% 45.1%64.4% 49.9% 0.17 10:39.6:50.4 27.154.2 leu:CaCl2:Na2SO4 Buchi 3.8 1.91.1 17.2% 37.5% 55.5% 56.1% 0.30 10:39.6:50.4 65-009-FLeucine:CaCl2:Na2SO4 Niro 2.5 2.2 1.4 1.5 60.1% 82.7% 88.6% 74.2% 0.3410.0:39.6:50.4 26.053.1 leucine:CaCl2:Na2SO4 Niro 4.2 2.0 1.5 3.3% 23.0%39.6% 52.0% 59.6% 50:22:28 27.114.4 leu:CaCl2:Na2SO4 Niro 4.7 1.8 1.93.8% 21.2% 44.6% 59.6% 59.6% 50:22:28 27.155.1 leu:CaCl2:Na2SO4 Buchi3.7 1.9 1.2 15.7% 42.9% 68.8% 47.6% 0.35 50:22:28 Calcium sulfate26.019.4 leu:CaSO4 50:50 Niro 4.1 2.1 1.4 11.9% 28.0% 56.0% 101.8%Carbonate 26.019.1 leu:CaCl2:NaCO3 Niro 3.4 1.9 1.7 9.6% 22.2% 35.9%46.3% 50:25.5:24.5 26.019.2 leu:CaCl2:NaCO3 Niro 2.7 1.8 1.4 10.6% 23.8%37.5% 51.0% 10:45.9:44.1 Lactate 26.041.4 leu:CaLact:NaCl Niro 5.0 1.99.7% 25.9% 46.6% 56.5% 50:36.8:13.1 27.183.2 Leu:CaLact:NaCl Buchi 3.71.8 1.1 24.9% 48.9% 62.7% 34.1% 50:48.4:1.6 27.185.1 Leu:CaLact:NaClBuchi 3.0 1.9 1.0 26.1% 53.7% 70.0% 44.8% 10:66.6:23.4 45.19.1leu:CaLact:NaCl Buchi 3.4 2.3 0.9 5.2% 12.8% 29.1% 50.3% 75.6% 0.7410:66.6:23.4 HP 45.76.1 leu:CaLact:NaCl Buchi 3.8 2.1 1.0 5.0% 8.6%20.9% 36.6% 78.5% 10:58.6:31.4 HP 45.78.1 leu:CaLact:NaCl Buchi 1.5 1.91.1 4.8% 30.6% 53.4% 62.9% 60.8% 10:58.6:31.4 HP 45.80.1 leu:CaLact:NaClBuchi 1.5 1.9 1.1 4.4% 30.3% 53.5% 63.8% 71.0% 10:58.6:31.4 HP 45.81.1leu:CaLact:NaCl Buchi 2.4 2.8 1.3 7.2% 19.3% 34.1% 44.3% 64.6%10:58.6:31.4 HP 68.70.1 leu:CaLact:NaCl Buchi 1.5 1.9 1.0 42.8% 63.2%67.8% 73.9% 10:58.6:31.4 HP 65-003-F Leucine:CaLact:NaCl Niro 1.5 2.51.1 1.1 43.4% 63.5% 69.7% 62.9% 0.69 10.0:58.6:31.4 Gluconate 27.184.1Leu:CaGluc:NaCl Buchi 3.4 2.1 1.0 35.0% 61.4% 76.3% 51.9% 50:49.15:0.8527.184.4 leu:CaGluc:NaCl Buchi 3.5 2.0 1.2 34.1% 60.7% 71.5% 46.3%50:42.35:7.65 27.184.2 Leu:CaGluc:NaCl Buchi 2.7 2.0 1.0 24.9% 52.2%64.2% 51.0% 10:88.5:1.5

Example 19

Pure calcium chloride was spray dried in the Labplant spray dryingsystem with an inlet temperature of 180° C. The liquid feed consisted of20 g/L solids concentration of calcium chloride dihydrate in D.I. water.Water condensed in the collection vessel as the calcium chloridedeliquesced and no powder could be collected. Pure calcium chloride wasdeemed too hygroscopic for spray drying from an aqueous solution withhigh water content in the exhaust drying gas. The liquid feed was thenchanged to 70% ethanol to reduce humidity in the exhaust gas, keepingthe solids concentration at 20 g/L, the inlet temperature at 200° C. andoutlet temperature at 69° C. Water still condensed in the collectionvessel and the powder looked wet. It was concluded that calcium chlorideis too hygroscopic to be spray dried without mixing with other salts orwith an excipient to reduce the calcium chloride content in the finalpowder.

Pure magnesium chloride was spray dried in the Labplant system with aninlet temperature of 195° C. and outlet temperature of 68° C. The liquidfeed consisted of 20 g/L solids concentration of magnesium chloridehexahydrate in D.I. water. The dry powder in the collection vessellooked wet and the median particle size measured on the HELOS/RODOSsystem was 21 microns. The liquid feed was then changed to 70% ethanolto reduce humidity in the exhaust drying gas, keeping the solidsconcentration at 50 g/L, the inlet temperature at 200° C. and an outlettemperature of 74° C. This magnesium chloride powder did not look wetand had a median volume particle size of 4 microns, but the powderappeared granular and had a fine particle fraction less than 5.6 micronsof 19%, indicating that the powder was not sufficiently respirable.

Example 20 Large, Porous Particles

x50 Spraytec FPF_TD FPF_TD Tapped (μm) GSD @ 1/4 0.5/4 dV50 Spraytecwater <3.4 um <5.6 um % Mass density Formulation Method @ 1 bar 1 barbar bar (μm) GSD % % % collected yield % (g/cc) leucine:Cacl2:NaCl Niro25.9 5.8 18.2% 29.0% 48.6% 43.2% 50:29.5:20.5 leucine:Cacl2:NaCl Niro12.2 6.3 35.4% 50:29.5:20.5 leu:CaCl2:Na2SO4 Niro 10.0 2.4 1.8% 5.0%16.5% 34.7% 84.8% 90:4.4:5.6 leu:CaLact:NaCl Buchi HP 22.4 4.4 4.9% 7.3%13.1% 72.0% 10:66.6:23.4 leu:CaCl2:Na2SO4 Buchi HP 21.2 3.0 13.2% 25.2%47.7% n/a 0.22 67.6:30:2.4

Table 33. Large Porous Particle Formulations Example 21 Stability

Dry powders were tested for in-use stability under extreme temperatureand humidity conditions (ICH, Climatic Zone XIV), defined as 30° C. and75% RH. Approximately 25 mg of Formulation I, Formulation II andFormulation III were filled into capsules. The capsules were left openedand then were placed in a stability chamber at the defined conditionsfor 15 and 30 minutes. The capsules were removed at the appropriatetime, closed and tested for aPSD using the collapsed 2-stage ACI and forgPSD using the Malvern Spraytec. Both tests were run at 60LPM for 2seconds. Each timepoint was repeated n=2. The results were compared withaPSD/gPSD data from the powder at room temperature and 25-30% RH.

All formulations (Formulation I, Formulation II and Formulation III)showed less than +/−5% change from the fine particle fraction of thetotal dose (FPFTD) less than 5.6 microns at standard conditions (22° C.,25-30% RH), after a 30 minute exposure to extreme temperature andhumidity conditions (30° C., 75% RH). For gPSD, Formulation I showed anincrease of approximately 30% after 30 minutes, while Formulation IIIremained mostly stable and Formulation II had a decrease in Dv50 ofapproximately 15% after 30 minutes.

While insignificant changes in aerosol properties of the threeformulations were seen upon exposure to 30° C., 75% RH for 30 minutes,changes in geometric particle size were more evident (FIGS. 31A and 31B). Formulation I (calcium citrate) particle size increased byapproximately 30%, while Formulation II (calcium lactate) particle sizedecreased by approximately 15%. Formulation III (calcium sulfate)particle size decreased, but not significantly.

Additional formulations tested were a calcium chloride powder (38.4%leucine, 30.0% calcium chloride, 31.6% sodium chloride) and thee calciumlactate powders using different excipients (lactose, mannitol,maltodextrin) matching the Formulation II formulation (10.0% excipient,58.6% calcium lactate, 31.4% sodium chloride).

After a 30 minute exposure to extreme temperature and humidityconditions (30° C., 75% RH), the maltodextrin (Formulation XIV) andmannitol formulations showed an overall change of less than +/−10%change from the fine particle fraction of the total dose smaller than5.6 microns at standard conditions (22° C., 25-30% RH). The calciumchloride powder and lactose formulation appeared affected with adecrease of over 50% and an increase of approximately 20%, respectively,in fine particle fraction of the total dose smaller than 5.6 microns.(FIG. 31C) For gPSD, the results were opposite, where the calciumchloride powder and the lactose formulation showed an overall change ofless than +/−10% change in Dv₅₀ after 30 minutes, while the mannitolformulation had an increase in Dv₅₀ of 30%-60% during the test. (FIG.32D) The maltodextrin formulation was not tested for change in Dv₅₀.

Example 22 Short-Term Stability at Room Temperature and 30% and 40% RH

Spray dried powders were kept at room temperature at approximately 30%and 40% RH for a period of one week and periodically tested for particlesize distribution. Size 3 HPMC capsules (Quali-V, Qualicaps, Whitsett,N.C.) were half filled with each dry powder. One sample was testedimmediately in the Spraytec (Malvern Instruments Inc., Westborough,Mass.), a laser diffraction spray particle sizing system where drypowders can be dispersed from an inhaler using the inhaler cell setup.Approximately 16 capsules were filled with each powder. Half of thecapsules were kept in the lab at controlled humidity and temperatureconditions (˜23-28% RH), while the other half were kept in the outsidelab at varying temperature and relative humidity (˜38-40% RH). Atspecific time points (t=1 hr, 2 hr, 4 hr, 24 hr, 48 hr, 1 week), onecapsule from the environmental controlled room and one from the outsidelab were tested on the Spraytec for volume particle size distribution.

Results for a selection of formulations containing 50% leucine and acombination of calcium chloride and the sodium salt indicated are shownin FIG. 32 and FIG. 33. The formulations containing calcium chloride andsodium chloride showed significant agglomeration after exposure tohigher humidity conditions. The acetate formulation had variable resultsat the initial time points. The sulfate, citrate and carbonateformulations demonstrated good relative stability over the test period.

Dry powder formulations containing calcium chloride and sodium chloridewere not stable when held at room temperature and 40% RH after an hourof exposure, while the acetate formulation also showed variable resultsin particle size. The sulfate and lactate powders increased slightly insize, while carbonate and citrate powders decreased slightly in size.Formulations containing only chloride and those containing acetate werenot deemed suitably stable for further study.

Example 23 Dry Powder Flow Properties

The flowability of Formulation I, II, III and XIV powders was alsoassessed using conventional methods in the art for the characterizationof powder flowability. The Flowability Index for each powder wasdetermined using a Flodex Powder Flowability Test Instrument (HansonResearch Corp., model 21-101-000). For any given run, the entire samplewas loaded using a stainless steel funnel aimed at the center of thetrap door hole in the cylinder. Care was taken not to disturb the columnof powder in the cylinder. After waiting ˜30 sec for the potentialformation of flocculi, the trap door was released while causing aslittle vibration to the apparatus as possible. The test was considered apass if the powder dropped through the trap door so that the hole wasvisible looking down through the cylinder from the top and the residuein the cylinder formed an inverted cone; if the hole was not visible orthe powder fell straight through the hole without leaving a cone-shapedresidue, the test failed. Enough flow discs were tested to find theminimum size hole the powder would pass through, yielding a positivetest. The minimum-sized flow disc was tested two additional times toobtain 3 positive tests out of 3 attempts. The flowability index (FI) isreported as this minimum-sized hole diameter.

Bulk and tap densities were determined using a SOTAX Tap Density Testermodel TD2. For any given run, the entire sample was introduced to atared 100-mL graduated cylinder using a stainless steel funnel. Thepowder mass and initial volume (V₀) were recorded and the cylinder wasattached to the anvil and run according to the USP I method. For thefirst pass, the cylinder was tapped using Tap Count 1 (500 taps) and theresulting volume V_(a) was recorded. For the second pass, Tap Count 2was used (750 taps) resulting in the new volume V_(b1). If V_(b1)>98% ofV_(a), the test was complete, otherwise Tap Count 3 was used (1250 taps)iteratively until V_(bn)>98% of V_(bn-1). Calculations were made todetermine the powder bulk density (d_(B)), tap density (d_(T)), HausnerRatio (H) and Compressibility Index (C), the latter two of which arestandard measures of powder flowability. “H” is the tap density dividedby the bulk density, and “C” is 100*(1−(bulk density divided by the tapdensity)). Skeletal Density measurement was performed by MicromeriticsAnalytical Services using an Accupyc II 1340 which used a helium gasdisplacement technique to determine the volume of the powders. Theinstrument measured the volume of each sample excluding interstitialvoids in bulk powders and any open porosity in the individual particlesto which the gas had access. Internal (closed) porosity was stillincluded in the volume. The density was calculated using this measuredvolume and the sample weight which was determined using a balance. Foreach sample, the volume was measured 10 times and the skeletal density(d_(S)) was reported as the average of the 10 density calculations withstandard deviation.

Results for these density and flowability tests are shown in Tables 34and 35. All four of the powders tested possess Hausner Ratios andCompressibility Indices that are described in the art as beingcharacteristic of powders with extremely poor flow properties (See,e.g., USP <1174>). It is thus surprising that these powders are highlydispersible and possess good aerosolization properties as describedherein.

TABLE 34 Bulk and tap densities and flow properties of FormulationI-IIII and XIV powders. FI d_(B) d_(T) Sample (mm) (g/mL) (g/mL) H CFormulation I 26 0.193 0.341 1.77 43.4% Formulation II 22 0.313 0.7222.31 56.7% Formulation III 18 0.177 0.388 2.19 54.3% Formulation XIV >340.429 0.751 1.75 42.9%

TABLE 35 Skeletal density measurements of powders Formulation I-II andXIV. Sample d_(S1) ± σ (g/mL) d_(S2) ± σ (g/mL) Formulation I 1.7321 ±0.0014 1.7384 ± 0.0042 Formulation II 1.6061 ± 0.0007 1.6074 ± 0.0004Formulation III 2.1243 ± 0.0011 2.1244 ± 0.0018 Formulation XIV 1.6759 ±0.0005 1.6757 ± 0.0005

USP <1174> mentioned previously notes that dry powders with a HausnerRatio greater than 1.35 are poor flowing powders. Flow properties anddispersibility are both negatively effected by particle agglomeration oraggregation. It is therefore unexpected that powders with Hausner Ratiosof 1.75 to 2.31 would be highly dispersible

Example 24 Water Content and Hygroscopicity

The water content of Formulation I, II, III and XIV powders wasdetermined via both thermogravimetric analysis (TGA) and Karl Fischeranalysis. Thermogravimetric analysis (TGA) was performed using a TAInstruments Q5000 IR thermogravimetric analyzer (New Castle, Del.).Sample was placed in an aluminum sample pan and inserted into the TGfurnace. The data acquisition and processing parameters are displayed oneach thermogram. Nickel and Alumel™ were used as the calibrationstandards. For TGA, the water content was determined from the loss ofmass of the samples upon heating to a temperature of 150° C. (for TGA,since the spray-drying solvent used was 100% water, it was assumed thatonly water was present as a volatile component in these powders). Arepresentative TGA thermogram for powder Formulation I is shown in FIG.34 Coulometric Karl Fischer (KF) analysis for water determination wasperformed using a Mettler Toledo DL39 KF titrator (Greifensee,Switzerland). Sample was placed in the KF titration vessel containingHydranal-Coulomat AD and mixed for 10 seconds to ensure dissolution. Thesample was then titrated by means of a generator electrode whichproduces iodine by electrochemical oxidation: 2 I−=>I₂+2e. Generally,one range-finding run and two replicates were obtained to ensurereproducibility. Summary data for powder water contents using thesemethods are shown in Table 36

TABLE 36 Water content data for FORMUALTIONS I, II, III and XIV via TGAand Karl fischer. Water Content Water Content Powder via TGA via KarlFischer Formulation I 4.9% 3.9% Formulation II 2.0% 2.0% Formulation III5.1% 4.6% Formulation XIV 2.2% 2.1%

A dynamic vapor sorption (DVS) step mode experiment was conducted tocompare the hygroscopicity and water uptake potential of Formulation I,II, III and XIV powders versus raw calcium chloride dihydrate, as wellas a 1:2 calcium chloride:sodium chloride control powder made viaspray-drying a formulation containing 38.4% leucine, 30% CaCl₂ and 31.6%NaCl (it was determined that 30 wt % was the highest loading level ofcalcium chloride that could be successfully incorporated into aspray-dried powder without undergoing deliquescence in the collectionvehicle immediately after spray-drying). With respect to the DVSoperating conditions, the powders were initially equilibrated at 0% RHthen exposed to 30% RH for 1 hour followed by exposure to 75% RH for 4hours. The mass % water uptake for each of the powders is shown in Table37. As can be seen in Table 37, both raw calcium chloride dihydrate andthe control powder were extremely hygroscopic, taking up approximately14 to 15% water upon exposure to 30% RH for 1 hour and taking up wellover 100% their mass in water after exposure to 75% RH. In contrast, theFormulation I, II, III and XIV powders took up less than 2.5% water uponexposure to 30% RH for 1 hour and from 14% to 33% water upon exposure to75% RH for 4 hours.

TABLE 37 % Change in mass due to water uptake after (i) 30% RH hold for1 hour and (ii) 75% RH hold for 4 hours via DVS. % Change in % Change inMass Due to Mass Due to Water Uptake Water Uptake after 30% RH after 75%RH Powder for 1 hr for 4 hrs CaCl₂*2H₂0 (raw) 13.7 146 CaCl₂-control15.3 124 Formulation I 1.68 14.7 Formulation II 1.27 28.3 FormulationIII 2.45 20.8 Formulation XIV 1.36 32.8

Example 25 Heat of Solution

Heats of solution were obtained upon dissolution of samples ofFormulations I through III in HBSS buffer in comparison to (i) a controlpowder comprised of 30% calcium chloride, 31.6% sodium chloride and38.4% leucine, (ii) raw calcium chloride dihydrate and (iii) rawleucine. As shown in Table 38, masses of Formulation I (PUR111), II(PUR113) and III (PUR112) powder containing equivalent moles of calciumion were tested for the calcium-containing samples. Results are shown inFIG. 35. As can be seen from the data shown in FIG. 35, Formulations Ithrough III resulted in significantly decreased heats of solution ascompared to both raw calcium chloride dihydrate and the control calciumpowder. Calcium chloride dihydrate is known to possess a largeexothermic heat of solution and to release a significant amount of heatupon contact with water. Under certain circumstances, such as when alarge quantity of calcium chloride dihydrate, or other salts that have alarge exothermic heat of solution, are rapidly dissolved a large amountof heat is released that can cause burns. Thus, there are safetyconcerns associated with contacting mucosal surfaces with calciumchloride dihydrate. These safety concerns can be alleviated by producingpowders, such as Formulations I through III which do not have largeexothermic heats of solution, and thus reduced potential for undesirableexothermic effects.

TABLE 38 Heat of solution data for Formulations I-III, a control powdercontaining calcium chloride, raw calcium chloride dihydrate and rawleucine. Powder Leucine CaCl2•2H2O CaCl2-control PUR111 PUR112 PUR113Lot # 65-017-F (−4) Spectrum 68-113-1 26-190-F 65-009-F 65-003-F Avg.St. Dev. Avg. St. Dev. Avg. St. Dev. Avg. Avg. Avg. St. Dev. Avg. St.Dev. g 0.032 0.000 0.036 0.001 0.090 0.001 0.077 0.000 0.068 0.000 0.0900.000 mmol* 0.244 0.001 0.242 0.000 0.242 0.000 0.242 0.000 0.243 0.0000.242 0.000 ΔT (deg. C.) 0.003 0.002 0.024 0.001 0.023 0.003 0.014 0.0020.012 0.003 0.009 0.002 Q (cal) 0.37 0.20 2.93 0.12 2.8 0.3 1.7 0.2 1.50.4 1.0 0.3 ΔH (kcal/mol)* −1.5 0.8 −12.1 0.4 −11.7 1.4 −6.9 1.0 −6.21.6 −4.3 1.1 ΔH (kJ/mol)* −6 4 −50.6 1.6 −49 6 −29 4 −26 7 −18 4 *mol Cafor all powders except leucine, which is in mol Leu

Example 26 In Vivo Pneumonia Model

Bacteria were prepared by growing cultures on tryptic soy agar (TSA)blood plates overnight at 37° C. plus 5% CO₂. Single colonies wereresuspended to an OD₆₀₀˜0.3 in sterile PBS and subsequently diluted 1:4in sterile PBS (˜2×10⁷ Colony forming units (CFU)/mL). Mice wereinfected with 50 μL of bacterial suspension (˜1×10⁶ CFU) byintratracheal instillation while under anesthesia.

C57BL6 mice were exposed to aerosolized liquid formulations in awhole-body exposure system using either a high output nebulizer or PariLC Sprint nebulizer connected to a pie chamber cage that individuallyholds up to 11 animals. Mice were treated with dry powder formulations(Table 39) 2 h before infection with S. pneumoniae. As a control,animals were exposed to a similar amount of 100% leucine powder.Twenty-four hours after infection mice were euthanized by pentobarbitalinjection and lungs were collected and homogenized in sterile PBS. Lunghomogenate samples were serially diluted in sterile PBS and plated onTSA blood agar plates. CFU were enumerated the following day.

Compared to control animals, calcium dry powder treated animalsexhibited reduced bacterial titers 24 hours after infection.Specifically, animals treated with a formulation comprised of calciumsulfate and sodium chloride (Formulation III) exhibited 5-fold lowerbacterial titers, animals treated with a formulation comprised ofcalcium citrate and sodium chloride (Formulation I) exhibited 10.4-foldlower bacterial titers, and animals treated with a formulation comprisedof calcium lactate and sodium chloride (Formulation II) exhibited5.9-fold lower bacterial titers. (FIG. 36)

TABLE 39 Formulations used to evaluate efficacy Ca:Na molar FormulationComposition ratio Formulation I 10.0% leucine, 35.1% calcium chloride,1:2 54.9% sodium citrate (Active with 12.7% calcium ion) Formulation III10.0% leucine, 39.6% calcium chloride, 1:2 50.4% sodium sulfate (Activewith 14.3% calcium ion) Formulation II 10.0% leucine, 58.6% calciumlactate, 1:2 31.4% sodium chloride (Active with 10.8% calcium ion)

The data presented herein show that divalent metal cationsalt-containing dry powders that are highly dispersible can bemanufactured and used to treat bacterial and viral infections.

Example 27 3 Month Refrigerated, Standard and Accelerated ConditionsStability Study

A 3 month physical stability study was conducted utilizingrepresentative samples of Formulations I through III filled into size 3HPMC capsules (Shionogi Qualicaps, Madrid, Spain) and placed at thefollowing conditions (i) 2-8° C. refrigerated storage, (ii) 25° C./60%RH, capsules stored under desiccant and (iii) 40° C./75% RH, capsulesstored under desiccant. FPF <5.6 and 3.4 as well as Dv50 (Spraytec) andwater content (Karl Fischer) were monitored out to a 3 month timepoint.As shown in Table 40, each of Formulations I through III displayed goodstability with respect to the assessed physical properties under each ofthese conditions.

TABLE 40 3 month stability study results for Formulations I-III.Formulation I (citrate) Formulation II (lactate) Formulation III(sulfate) Condition Time FPF <3.4 FPF <5.6 Spraytec FPF <3.4 FPF <5.6Spraytec FP F<3.4 FPF <5.6 Spraytec (° C./% RH) (mo) um um (um) H2O umum (um) H2O um um (um) H2O Time zero 0 50% 63% 3.1 6% 42% 61% 1.8 4% 55%73% 3.1 5% 25 C./ 1 47% 68% 1.5 7% 42% 60% 2.0 4% 56% 74% 3.6 6% 60% RH3 45% 68% 3.5 7% 42% 61% 1.2 4% 57% 73% 2.4 6% (capsules + desiccant) 40C./ 0.5 43% 66% 5.3 8% 39% 58% 1.8 6% 51% 67% 2.9 6% 75% RH 1 43% 65%2.0 7% 40% 58% 3.0 4% 56% 70% 3.9 5% (capsules + 3 46% 68% 3.3 7% 47%61% 1.5 4% 45% 64% 2.5 5% desiccant) 2-8 C. 3 46% 60% 2.4 5% 43% 63% 1.32% 56% 76% 2.3 5%

The content of each of the patents, patent applications, patentpublications and published articles cited in this specification areherein incorporated by reference in their entirety.

1. A respirable dry powder comprising respirable dry particles thatcomprise a divalent metal cation salt, wherein the divalent metal cationsalt provides divalent metal cation in an amount of about 3% to about 5%by weight of the dry particle, and the respirable dry particles thatcomprise a divalent metal cation salt have a volume median geometricdiameter (VMGD) of about 5 microns or less, a tap density of greaterthan 0.4 g/cubic centimeter (cc) and are further characterized by acapsule emitted powder mass (CEPM) of at least 80% when emitted from apassive dry powder inhaler that has a resistance of about 0.036sqrt(kPa)/liters per minute (LPM) under the following conditions: aninhalation energy of 1.15 Joules at a flow rate of 30 LPM using a size 3capsule that contains a total mass of 25 mg, said total mass consistingof the respirable dry particles that comprise a divalent metal cationsalt, and wherein the VMGD of the dry particles emitted from the inhaleris 5 microns or less.
 2. The respirable dry powder of claim 1, whereinsaid divalent metal salt is selected from the group consisting of amagnesium salt, and a barium salt.
 3. The respirable dry powder of claim1, wherein the divalent metal cation salt is a calcium salt.
 4. Therespirable dry powder of claim 3, wherein said calcium salt is selectedfrom the group consisting of calcium citrate and calcium sulfate.
 5. Therespirable dry powder of claim 1, wherein the divalent metal cation saltis a magnesium salt.
 6. The respirable dry powder of claim 1, whereinthe respirable dry particles that comprise a divalent metal cation saltfurther comprise at least one pharmaceutically acceptable excipient. 7.The respirable dry powder of claim 6, wherein the at least one excipientis present in an amount of about ≦50% by weight and comprises leucine.8. The respirable dry powder of claim 6, wherein the at least oneexcipient is present in an amount of about ≦50% by weight and comprisesmaltodextrin or mannitol.
 9. The respirable dry powder of claim 1,wherein the respirable dry particles that comprise a divalent metalcation salt further comprises a monovalent salt.
 10. The respirable drypowder of claim 9, wherein the monovalent salt is a lithium salt or apotassium salt.
 11. The respirable dry powder of claim 9, wherein themonovalent salt is a sodium salt.
 12. A method for treating arespiratory disease comprising administering to the respiratory tract ofa patient in need thereof an effective amount of a respirable dry powderof claim
 1. 13. A method for treating an acute exacerbation of arespiratory disease comprising administering to the respiratory tract ofa patient in need thereof an effective amount of a respirable dry powderof claim 1.